Optoelectronic tweezers for microparticle and cell manipulation

ABSTRACT

An optical image-driven light induced dielectrophoresis (DEP) apparatus and method are described which provide for the manipulation of particles or cells with a diameter on the order of 100 μm or less. The apparatus is referred to as optoelectric tweezers (OET) and provides a number of advantages over conventional optical tweezers, in particular the ability to perform operations in parallel and over a large area without damage to living cells. The OET device generally comprises a planar liquid-filled structure having one or more portions which are photoconductive to convert incoming light to a change in the electric field pattern. The light patterns are dynamically generated to provide a number of manipulation structures that can manipulate single particles and cells or group of particles/cells. The OET preferably includes a microscopic imaging means to provide feedback for the optical manipulation, such as detecting position and characteristics wherein the light patterns are modulated accordingly.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application Ser.No. 60/561,587 filed on Apr. 12, 2004, incorporated herein by referencein its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.442521-WM-22622/NCC2-1364, awarded by NASA. The Government has certainrights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable

NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION

A portion of the material in this patent document is subject tocopyright protection under the copyright laws of the United States andof other countries. The owner of the copyright rights has no objectionto the facsimile reproduction by anyone of the patent document or thepatent disclosure, as it appears in the United States Patent andTrademark Office publicly available file or records, but otherwisereserves all copyright rights whatsoever. The copyright owner does nothereby waive any of its rights to have this patent document maintainedin secrecy, including without limitation its rights pursuant to 37C.F.R. § 1.14.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains generally to cell and microparticlemanipulation, and more particularly to optoelectronic tweezers (OET).

2. Description of Related Art

The ability to manipulate biological cells and micrometer scaleparticles plays an important role in many biological and colloidalscience applications. However, conventional manipulation techniques,including optical tweezers, electrokinetic forces (electrophoresis,dielectrophoresis (DEP), traveling-wave dielectrophoresis), magnetictweezers, acoustic traps, and hydrodynamic flows, cannot simultaneouslyachieve high resolution and high throughput.

DEP is a well established technique that has been widely used tomanipulate micrometer and sub-micrometer particles as well as biologicalcells. Traveling-wave dielectrophoresis (TWD) is particularly attractivefor high throughput cell manipulation without external liquid pumping.The traveling electric field produced by multi-phase alternating current(AC) bias on a parallel array of electrodes levitates and transportsmany particles simultaneously. However, the TWD cannot resolveindividual particles. Recently, a programmable DEP manipulator withindividually addressable two-dimensional electrode array has beenrealized using complementary metal-oxide-semiconductor (CMOS) integratedcircuit (IC) technology. Parallel manipulation of a large number (i.e.,approximately 10,000) of individual cells was demonstrated. The CMOS DEPmanipulator has two potential drawbacks. The need of on-chip ICincreases the cost of the chip, making it less attractive for disposableapplications. The trap density (i.e., approximately 400 sites/mm²) isalso limited by the size of the control circuits.

Consequently, the use of electrokinetic forces and similar mechanismsprovide high throughput, but lack the flexibility or the spatialresolution for controlling individual cells, or groups of cells. Inaddition, these techniques require structures formed through numerouslithographic steps.

Optical tweezers, however, offer high resolution for trapping singleparticles, yet provide limited manipulation area due to tight focusingrequirements. The optical tweezers use direct optical force for themanipulating purpose, and require highly focused coherent light sourcesused with an objective lens having a high numerical-aperture (N. A.)value and a small field of view. To generate multiple optical traps orspecial optical patterns, it also requires techniques such asholography. These techniques require intense calculation for creatingeven simple optical patterns.

Accordingly a need exists for a particle and cell manipulation apparatusand method which provides parallel processing capability while stillproviding selectivity down to the single particle level. The presentinvention fulfills those needs, as well as others, and provides formanipulation of particles and cells at low light levels without the needof complex lithography or 3D beam control.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to performing particle and cellmanipulation using optical image-driven light induced dielectrophoresis(DEP). The term “particle” will be used herein to referencemicroparticles, nanoparticles, cells, and other organic and inorganicmaterial having a diameter generally between a few nanometers up to theorder of approximately 100 μm. The techniques allow for the use ofmoderate intensity incoherent light sources, which create dynamicpatterns that can be controlled in response to image detection andprocessing of actual particle composition and position.

In accordance with one embodiment of the invention, an opticalimage-driven dielectrophoresis apparatus and method is described forpatterning electric fields on a photoconductive surface for manipulatingsingle particles, or collections of particles. A wide variety of lightsources can be utilized, such as incoherent light, and single ormultistage manipulation of particles can be readily achieved.

One embodiment comprises optoelectronic tweezers (OET) configured forcell and microparticle manipulation using optical control. The OETpermits functions such as cell trapping, repelling, collecting,transporting, and sorting of cells and microparticles by usingsequentially projected images controlled by a spatial light modulator(e.g., microdisplay or DMD mirrors, and so forth). With opticalactuating power as low as 1 mW optical manipulation can be performedusing incoherent lightly focused light and a direct image projectionsystem.

In one embodiment, dynamic DMD-driven optoelectronic tweezers performdynamic manipulation of microscopic particles using a DMD producedprojection image. Single-particle trapping and movement (up to 40μm/sec) via optically-induced dielectrophoresis were observed in thisembodiment.

Another embodiment is described in which dynamic array manipulation ofparticles and microparticles is performed using optoelectronic tweezers.One demonstration details the individual trapping of polystyreneparticles with 20 μm and 45 μm diameters trapped by light patternsgenerated by a digital light projector with digital micromirror device(DMD). Self-organization and individual addressing of the particles aredemonstrated. Movement of 45 μm polystyrene particles is measured to be35 μm/sec (a force of 15 pN).

Another embodiment provides for the manipulation of live red and whiteblood cells with optoelectronic tweezers. Optically-induceddielectrophoresis is enabled within the optoelectronic tweezers (OET) tomanipulate live mammalian cells demonstrated by concentrating bovine redblood cells in solution. A spatial light modulator and an incoherentlight source integrated in combination with the OET provide the abilityto easily create reconfigurable, complex manipulation patterns. Thiscapability is also demonstrated in the patterning of human white bloodcells into complex patterns.

Another embodiment provides for using light induced dielectrophoresis tooptically trap and transport micro particles with optical power in themicrowatt range. This embodiment comprises two pattern-less(unpatterned) surfaces: a bottom glass substrate coated withphotoconductive material and a top transparent indium-tin-oxide (ITO)glass. To achieve optical trapping, the liquid-immersed micro-particlesare sandwiched between these two surfaces and an AC electric bias issupplied. A 633 nm He—Ne laser focused by a 40× objective lens is usedto transport the particles. Negative dielectrophoretic trapping isdemonstrated and the experimental results show that optical beams withpower as low as 1 μW are sufficient to transport 25 μm diameter latexparticles at a speed of 4.5 μm/sec. The transport speed increases withhigher optical power. A maximum speed of 397 μm/sec is observed at 100μW.

In this embodiment, an optical sorting mechanism is described based on adynamic electric field patterned by the scanning optoelectronic tweezers(OET). The sorting mechanism is based on the force balance between thehydrodynamic viscous force and the dynamic light-induceddielectrophoretic force. Randomly distributed particles with differentsizes are sorted out and positioned in size-dependent deterministicpositions relative to a line-shaped scanning laser beam. A 240 μm laserbeam moving at a speed of 10 μm/sec can sort polystyrene beads withdiameters of 5 μm, 10 μm, and 20 μm to relative positions of 17 μm, 29μm, and 60 μm from the beam center.

This embodiment also provides for moving toward an all opticallab-on-a-chip system requiring optical manipulation tools for bothmicroparticles and microfluids. Although optical tweezers are importantfor manipulating cells or particles, they are not effective in handlingmicrofluid. The typically high optical power requirements have alsolimited the applicability in high throughput bioanalysis system. In thisembodiment two novel mechanisms are demonstrated: (1) optoelectrowetting(OEW) for handling microdroplets, and (2) optoelectronic tweezers (OET)for optical manipulation of microscopic particles with low optical poweractuation. Instead of using direct optical force, both mechanisms relyon light induced electrical force for optical manipulation.Optoelectrowetting (OEW) enables control of microfluids in droplet formby optical beams and is based on light induced electrowetting, whichchanges surface tension at solid-liquid interface at illuminated area.It is realized by integrating a layer of photoconductive material withelectrowetting electrodes. By programming the illumination pattern, wehave successfully demonstrated various functions for droplets, such asmoving, splitting, and merging. A 100 μL droplet was transported at aspeed of 785 μm/sec by an optical beam with an optical power of 100 μW.

Optoelectronic tweezers (OET) manipulate cells or particles based onlight induced dielectrophoresis (DEP). Trapping or repelling ofmicroscopic particles is achieved with a light intensity of 2 W/cm²,which is five orders of magnitudes lower than that required by opticaltweezers (approximately 105 W/cm² to 107 W/cm²). The liquid containingcells or particles is sandwiched between a photosensitive surface and atransparent ITO glass, with an AC bias between them. When the laser beamis focused on the photosensitive layer, it creates a virtual electrodeon the illuminated area, resulting a nonuniform electric field at theaqueous layer. Cells or particles in the liquid layer are polarized bythis non-uniform electric field and driven by the DEP force. The forcecould be attractive or repulsive, depending on the dielectric propertiesof the particles and the bias frequency. Using OET, we have demonstratedconcentration of polystyrene particles and live E. coli cells using anoptical power less than approximately 10 μW.

According to another embodiment of the invention, an apparatus formanipulating cells or particles by light induced dielectrophoresis (DEP)comprises: (a) a first surface and a second surface configured forretaining a liquid comprising particles or cells to be manipulated; (b)at least one photoconductive area on the first or the second surfaceconfigured for conversion of received light to a local electric field inthe vicinity of the received light; and (c) means for directing lightpatterns for receipt on the photoconductive area to selectively repel orattract particles or cells in response to the induced local electricfield. The light pattern directing means preferably comprises a lightsource configured for generating two-dimensional light patterns.

In accordance with another embodiment of the invention, anoptoelectronic tweezers (OET) apparatus for manipulating cells andparticles using optical image-driven light induced dielectrophoresis(DEP) over a two-dimensional area comprises: (a) a first surface andsecond surface having sufficient separation for retaining a liquid whichcontains particles, or cells, to be manipulated; (b) at least onephotoconductive area on the first or second surface which is configuredfor conversion of optical energy to an electric field in thephotoconductive area to create a local electric field, or virtualelectrode, in the vicinity of the received light; and (c) means fordynamic optical image positioning on the at least one photoconductivearea to generate moving virtual electrode patterns for manipulating thepositioning of particles or cells using light-induced dielectrophoresis(DEP).

Another embodiment of the invention provides an apparatus formanipulating cells or particles by light induced dielectrophoresis(DEP), the apparatus comprising: (a) a first surface and a secondsurface configured for retaining a liquid comprising particles or cellsto be manipulated; (b) at least one photoconductive area on the first orthe second surface configured for conversion of received light to alocal electric field in the vicinity of the received light; and (c) alight source to provide the light received by the photoconductive area,wherein the local electric field selectively repels or attractsparticles or cells.

The light source preferably comprises an optical projection systemconfigured for generating two dimensional light patterns, such as in theform of image sequences or streams upon the photoconductive area.

In a further embodiment of the invention, an optoelectronic tweezers(OET) apparatus for manipulating cells and particles using opticalimage-driven light induced dielectrophoresis (DEP) over atwo-dimensional area, comprises: (a) a first surface and second surfacehaving sufficient separation for retaining a liquid which containsparticles, or cells, to be manipulated; (b) at least one photoconductivearea on the first or second surface which is configured for inducing anelectric field, thereby creating a virtual electrode, in the vicinity ofthe received light (or similarly converting optical energy to anelectric field) and (c) an optical projector configured for generatingdynamic sequential two-dimensional light patterns onto thephotosensitive surface thereby inducing dynamic localized electricfields for DEP manipulation of particles or cells.

The optical projector, or similar means of dynamically projecting lightimages is preferably directed at the OET through a lens assembly, sothat a sequence of images can be formed onto the photoconductive area.In one preferred embodiment of the invention electrodes are coupled tothe first and second surfaces so that a bias signal can be applied tothe liquid with the contained particles or cells.

According to one aspect of the invention, a means is provided to performmicroscopic imaging of the particles and/or cells and to register theposition, and optionally the characteristics, of particles and/or cellsto provide feedback for controlling optical image positioning anddynamic image movement.

Another embodiment of the invention provides an optoelectronic tweezers(OET) apparatus for manipulating cells and particles (typically on theorder of 100 μm diameter or less) using optical image-driven lightinduced dielectrophoresis (DEP) over a two-dimensional area, comprising:(a) a first surface and second surface having sufficient separation forretaining a liquid which contains particles and/or cells to bemanipulated; (b) at least one photoconductive area on the first orsecond surface which is configured for conversion of optical energy toan electric field in the photoconductor to create a local electricfield, or virtual electrode, in the vicinity of the received light; (c)an optical projector coupled to a lens assembly configured forgenerating dynamic sequential images (a sequence of light patterns)through the lens assembly onto the photosensitive surface for creatingdynamic localized electric fields for the DEP manipulation of nearbyparticles and/or cells.

The first surface and second surface preferably form a continuous filmupon which DEP manipulation is performed in response to images receivedfrom the optical projector. In this way lithographic patterning withconductive electrodes is not necessary for performing DEP manipulation.

In one mode of the invention at least one electrode is coupled to eachof the first and second surface so that a bias signal can be applied tothe liquid with its particles and/or cells. The liquid preferablycomprises a conductive or semiconductive fluid. Typically a thindielectric layer is joined to the interior surface of the electrodes andconfigured to have an impedance that is less than the impedance acrossthe liquid.

In a preferred mode of the invention a microvision-based patternrecognition subsystem is configured for controlling the output of theoptical projector in response to registering the position of, andoptionally the characteristics of, particles and/or cells as determinedfrom microscopic imaging. The characteristics can comprise anythingwhich is directly detectable by the microscopic imaging system or whichcan be determined in response to detecting changes in the directcharacteristics over time. By way of example the characteristics caninclude size, color, shape, texture, viability, motility, conductivity,permeability, capacitance and response to changes in the environment ofthe particle or cell.

Another embodiment of the invention is an apparatus for manipulatingcells by light induced dielectrophoresis (DEP), the apparatuscomprising: (a) a first surface and a second surface configured forretaining a liquid comprising cells to be manipulated; (b) at least onephotoconductive area on the first or the second surface configured forinducing a local electric field in response to received light; (c) alight source to provide the light received by the photoconductive area,wherein the local electric field induced by the light selectively repelsor attracts cells and wherein the light received is of sufficiently lowoptical intensity that it does not damage the cells being manipulated inthe apparatus. In one preferred mode the embodiment further comprises amicroscopic imaging subsystem configured for controlling the output ofthe light source in response to registering the position of, andoptionally the characteristics of cells within the apparatus.

Another embodiment provides a method of manipulating particle orcellular objects retained within a liquid, the method comprising thesteps consisting essentially of: (a) confining the liquid comprising theparticle objects or cellular objects within a structure comprising atleast a first and second surface; (b) applying a bias voltage to theliquid by applying a bias signal to electrodes coupled to the first andsecond surfaces; (c) directing light to a photoconductive portion of thestructure, wherein the light induces a local electric field in thevicinity of the portion receiving light thereby dielectrophoreticallyrepelling or attracting the particles or cells.

Another embodiment provides a method of manipulating biological objectsretained within a liquid, the method comprising: (a) confining theliquid comprising the particle objects or cellular objects within astructure comprising at least a first and second surface; (b) applying abias voltage to the liquid by applying a bias signal to electrodescoupled to the first and second surfaces; (c) generating control signalsin response to registering the characteristics and positions ofbiological objects within the structure; (d) directing light in responseto the control signals upon a photoconductive portion of the structureto induce a local electric field in the vicinity of the received lightto dielectrophoretically repel or attract cellular objects, wherein thelight is of sufficiently low intensity that live cells being manipulatedby the method remain alive and viable.

Another embodiment of the invention generally provides a method ofdynamically manipulating particle and cellular objects retained within aliquid, comprising: (a) confining a liquid containing particle objects,and/or cellular objects within a structure having at least a first andsecond surface; (b) applying a bias voltage to the liquid by applying abias signal to electrodes coupled to the first and second surfaces; (c)focusing a light pattern on a photoconductive portion of the firstsurface and/or second surfaces, so that the optical energy of the lightis converted to a local electric field to create a virtual electrode inthe vicinity of the received light; and (d) dynamically positioning thelight pattern in response to feedback received from registering theposition, and optionally characteristics, of the particles and/or cells.

Another embodiment of the invention provides a method of dynamicallysorting cells retained within a liquid, comprising: (a) confining aliquid contains cells within a structure having at least a first andsecond surface; (b) applying a bias voltage to the liquid by applying abias signal to electrodes coupled to the first and second surfaces; (c)generating control signals in response to registering thecharacteristics and positions of cells within the structure anddetermining into which category cells are to be sorted; (d) directinglight, of sufficient low intensity to prevent cellular damage, inresponse to the control signals upon a photoconductive portion of thestructure to induce a local electric field in the vicinity of thereceived light to dielectrophoretically repel or attract the cells; and(e) sequentially directing light in response to the control signals tomove categorized cells into different sort groups within the structureor for conveyance outside of the structure.

A still further embodiment of the invention provides a method ofdynamically sorting particles or cells retained within a liquid,comprising: (a) confining a liquid containing particles or cells withina structure having at least a first and second surface; (b) applying abias voltage to the liquid by applying a bias signal to electrodescoupled to the first and second surfaces; and (c) directing a movingpattern of light across a photoconductive portion of the structure toinduce a local electric field in the vicinity of the pattern todielectrophoretically repel particles or cells displacing them from thepattern according to their relative size.

Embodiments of the present invention can provide a number of beneficialaspects which can be implemented either separately or in any desiredcombination without departing from the present teachings.

An aspect of the invention is to provide an apparatus and method formanipulating cells and particles using optical image-driven lightinduced dielectrophoresis (DEP) within a generally planar liquid-filledstructure.

Another aspect of the invention is performing optical image-driven lightinduced DEP over a large two-dimensional area adjacent to a fluidcontaining single particles and/or cells, collections of particlesand/or cells, or a combination thereof.

Another aspect of the invention is performing optical image-driven lightinduced DEP in which single particles and/or cells, or particle groupsand/or cell groups, can be manipulated in parallel (simultaneously).

Another aspect of the invention is performing optical image-driven lightinduced DEP wherein any of the particles or groups of particles can bemanipulated in any desired direction within the apparatus as they arenot constrained by a physical electrode structure.

Another aspect of the invention allows for performing opticalimage-driven light induced DEP using conventional materials andprocessing techniques.

Another aspect of the invention allows for performing opticalimage-driven light induced DEP on particles which may beelectrostatically neutral.

Another aspect of the invention allows for performing opticalimage-driven light induced DEP in an optoelectric tweezers device (OET)which achieves high resolution and high throughput simultaneously.

Another aspect of the invention allows for performing opticalimage-driven light induced DEP in an optoelectric tweezers device (OET)which creates dynamic electric fields to manipulate particle positioningwithout the assistance of fluidic flow.

Another aspect of the invention allows for performing opticalimage-driven light induced DEP in an optoelectric tweezers device (OET)which is capable of manipulating the position of particles and cells atless than approximately 10 μW which is about 1/100,000^(th) of theoptical energy level required by conventional optical tweezers.

Another aspect of the invention allows for an optoelectric tweezersdevice (OET) in which tight optical focusing is not required therebyallowing manipulation over a maximum area on the order of one squaremillimeter (1 mm²), or larger up to, such as 1.3 mm×1.0 mm which is manyorders of magnitude larger than that which is achievable usingconventional optical tweezers.

Another aspect of the invention allows for performing opticalimage-driven light induced DEP in an optoelectric tweezers device (OET)in combination with continuous optical electrowetting techniques (COEW).

Another aspect of the invention is an OET device having a first surfaceand second surface separated by chamber walls and configured forretaining a liquid which contains particles, or cells, beingmanipulated.

Another aspect of the invention is an OET device having electrodes onthe first and second surface upon which a biasing current and/or fieldcan be applied through and/or across the retained liquid.

Another aspect of the invention is an OET device having at least onephotosensitive/photoresponsive surface which creates a local electricfield in response to received light, therein creating virtual electrodesfor manipulating particles, cells, and the like at low optical powerlevels.

Another aspect of the invention is an OET device having first and secondsurfaces formed as a continuous film, wherein lithographic patterning ofthe surface is not necessary for practicing the invention.

Another aspect of the invention is an OET device having surfaces ofamorphous and/or micro/nano-crystalline semiconductor materials,amorphous Si, or organic photoconductor materials, used with or withoutdielectric layers, such as silicon nitride, silicon dioxide, and soforth.

Another aspect of the invention is an OET device having thin dielectriclayers with a lower impedance than the liquid retained in the OETdevice.

Another aspect of the invention is an OET device using a single ordouble-sided photosensitive surface in various combinations with aconductive surface, non-conductive surface, or no opposing surface (openstructure).

Another aspect of the invention is an OET device configured forimplementing traps, combs, sorters, concentrators, loops, conveyers,joints, particle channels, wedges, sweepers, which can be implementedseparately, in arrays of manipulation elements, and combinations andsequences of manipulation elements and so forth.

Another aspect of the invention is an OET device wherein the surfaceintegrates with microfluidic devices, such as channels, cavities,reservoirs, and pumps.

Another aspect of the invention is an OET device for implementing a combdevice for separating particles, cells, and other micro/nano-particlesin response to their size.

Another aspect of the invention is an OET device which can be biasedwith AC of a desired frequency, and/or DC biasing.

Another aspect of the invention is an OET device in which the frequencyof the AC bias determines whether particles, cells, and the like areattracted or repelled by the patterned light.

Another aspect of the invention is an OET device in which a microscopicimaging means operates in combination with the dynamic light patterningdevice so that patterns are created in response to the actualpositioning of particles, cells, and the like within the OET device.

Another aspect of the invention is an OET device in combination with amicroscopic imaging device which is configured to provide feedback tothe OET device during the characterization and positioning of particles.

Another aspect of the invention is an OET device in which themicroscopic imaging means is configured for analyzing the actualpositioning and composition of particles, cells, and so forth andcontrolling the generation of light output sequences for moving,collecting, and/or dispersing the particles, cells, and so forth inresponse to actual positions detected by the imaging means.

Another aspect of the invention is an OET device in which a conductiveor semiconductive fluid is retained in the device.

Another aspect of the invention is an OET device which is configured formanipulating particles and cells in response to light patterns, and inparticular dynamic lighting patterns.

Another aspect of the invention is an OET device in which dynamiclighting patterns are generated to sequentially move particles, cellsand so forth, in response to the light pattern motion.

Another aspect of the invention is an OET device in which the lightingpatterns are generated by a laser or more preferably a low-intensityincoherent light source (i.e., halogen, LEDs, and so forth).

Another aspect of the invention is an OET device in which the use oflow-intensity lighting is made possible by the conversion of opticalenergy to an electric field in the photoconductor.

Another aspect of the invention is an OET device configured for sortingparticles, or biological cells, in response to differences in viability(i.e., dead or alive), internal conductivity, size, color, shape,texture, response to changes in the aqueous environment, and similardistinguishing characteristics.

Another aspect of the invention is an OET device configured for sortingbiological cells in response to differences in membrane properties(e.g., permeability, capacitance, and so forth), internal conductivity,and the like.

Another aspect of the invention is an OET device in which the use oflow-intensity lighting allows for manipulating biological objectswithout loss of viability from photodamage (“opticution”) which ariseswhen using conventional optical tweezers.

Another aspect of the invention is an OET device in which the use oflow-intensity lighting is made possible by the conversion of opticalenergy to an electric field in the photoconductor.

Another aspect of the invention is an OET device in which the lightsource is patterned by a spatial light modulator, or similar form oflight modulator.

Another aspect of the invention is an OET device on which the lightpatterns are varied in response to magnification or demagnification.

A still further aspect of the invention is an OET device for use inbiological analysis, cell manipulation, colloidal assembly, particlesorting, particle assembly, and so forth.

Further aspects of the invention will be brought out in the followingportions of the specification, wherein the detailed description is forthe purpose of fully disclosing preferred embodiments of the inventionwithout placing limitations thereon.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The invention will be more fully understood by reference to thefollowing drawings which are for illustrative purposes only:

FIG. 1 is a perspective schematic view of optoelectronic tweezers (OET)according to an aspect of the present invention for manipulatingmicroscopic particles sandwiched between structure layers biased with anAC signal.

FIGS. 2A-2D are images of particle manipulation using particle trapsaccording to an aspect of the present invention, showing particles beingtrapped in a am array.

FIGS. 3A-3D are images of an integrated virtual optical machineaccording to an aspect of the present invention with a sorter, conveyor,joints, and a wedge for sorting microparticles.

FIGS. 4A-4D are images of optical sorting of live and dead cells usingthe OET according to an aspect of the present invention.

FIG. 5 is a schematic of an OET device according to an aspect of thepresent invention, showing the use of light-defined virtual electrodeswhich generate non-uniform electric fields in the liquid layer.

FIG. 6 is a block diagram of an OET-based cell manipulation systemaccording to an aspect of the present invention, showing the use of aprogrammable spatial light modulator for generating the desired opticalimage.

FIG. 7A is a perspective view of OET device structure according to anaspect of the present invention, showing particles retained in solutionbetween a first layer and an optically responsive second layer.

FIG. 7B is a schematic of an experimental OET setup according to anaspect of the present invention, showing the OET device of FIG. 7Areceiving a modulated and directed light source.

FIG. 8 is a 3-D graph of electric-field distribution for a singleparticle ring trap according to an aspect of the present invention.

FIGS. 9A-9C are images of particle manipulation according to an aspectof the present invention, shown using a dynamic line cage with two anglesections (forming a square in FIG. 9A) which contain the particles insuccessively smaller regions.

FIGS. 9D-9F are images of particle trapping according to an aspect ofthe present invention, showing trapping and moving a single particlewhile repelling particles outside of the selection area (ring).

FIG. 10A is an image of a single-particle OET trap according to anaspect of the present invention, shown retaining a 45 μm polystyrenesphere.

FIG. 10B is a graph of electric field distribution for the OET of FIG.10A.

FIG. 11 is a perspective view of an OET device according to an aspect ofthe present invention, showing particles in the liquid buffer retainedbetween the top and bottom layers of the OET.

FIG. 12 is a block diagram of an experimental OET setup according to anaspect of the present invention, showing light controlled from a PCdirected using DMD onto the OET device.

FIGS. 13A-13B are images of self-organization of microparticles into anarray configuration according to an aspect of the present invention.

FIGS. 14A-14B are images of single-particle manipulation within an OETarray according to an aspect of the present invention.

FIGS. 15A-15D are images of particle array flushing within an OET arrayaccording to an aspect of the present invention.

FIG. 16 is an image of an array of single particles trapped in an OETarray according to an aspect of the present invention.

FIGS. 17A-17D are images of dynamic rearrangement of differently sizedparticles according to an aspect of the present invention.

FIG. 18A is a schematic diagram of a microvision-based automatic opticalmanipulation system according to an aspect of the present invention.

FIG. 18B is a schematic of the OET device shown in the system of FIG.18A.

FIGS. 19A-19D is an illustration of steps according to an aspect of thepresent invention for arranging particles into any desired pattern.

FIGS. 20A-20D are images from a particle recognition system and a graphof recognition percentage according to an aspect of the presentinvention.

FIGS. 21A-21B are graphs of electric field distribution induced by asingle optical ring pattern according to an aspect of the presentinvention.

FIGS. 22A-22C are images of microvision-based automatic opticalmanipulation of microscopic particles according to an aspect of thepresent invention.

FIG. 23 is a schematic of an OET device according to an aspect of thepresent invention, showing modification of the electric-field patternswithin the particle-laden liquid.

FIG. 24 is a schematic of an experimental OET device setup according toan aspect of the present invention, shown using a modulated laser lightsource to direct optical particle manipulation patterns.

FIGS. 25A-25D are images of OET particle manipulation using acombination of optical input and AC biasing according to an aspect ofthe present invention.

FIG. 26 is an image of OET particle manipulation according to an aspectof the present invention, shown in the process of forming the letters“UC” with human white blood cells.

FIG. 27A is a perspective view of an OET device according to an aspectof the present invention, showing a focused beam directed through aliquid to a photoresponsive material.

FIG. 27B is a schematic of the operation of the OET of FIG. 27A inresponse to one mode of induced dielectrophoresis.

FIG. 28 is a graph of the relationship between particle speed andoptical power for an OET according to an aspect of the presentinvention.

FIGS. 29A-29B are images of using an OET to focus/concentrate multipleparticles according to an aspect of the present invention.

FIGS. 30A-30C are schematics of an OET device according to an aspect ofthe present invention, showing how different sized particles areorganized in response to an optical wave induced electric field.

FIG. 31 is a block diagram of an OET optical setup according to anaspect of the present invention, showing a laser illumination sourcewith underside microscopic imaging.

FIGS. 32A-32D are images of OET-based size sorting according to anaspect of the present invention, showing size sorting of particles ofapproximately 10 μm and 20 μm.

FIGS. 33A-33B are images of OET-based size sorting for particles in arange of sizes according to an aspect of the present invention.

FIG. 34 is a graph of distance versus speed for the different particlesizes demonstrated according to an aspect of the present invention.

FIG. 35 is a flow diagram comparison of the energy transfer pathsaccording to different optical manipulation methods.

FIGS. 36A-36B are schematics of an OEW device according to an aspect ofthe present invention, showing the change in droplet characteristicswhen the device is illuminated.

FIG. 37A is a schematic of droplet transport on a continuous OEW surfaceaccording to an aspect of the present invention.

FIG. 37B is a schematic of an equivalent electrical circuit for the OEWof FIG. 37A.

FIG. 37C is a perspective view of the layer structure of the COEWsurface according to an aspect of the present invention.

FIGS. 38A-38D are images of microdroplet transport utilized COEWaccording to an aspect of the present invention.

FIG. 39 is a schematic of an OET device according to an aspect of thepresent invention, showing particle containing liquid retained within astructure having a photoconductive surface for converting optical energyto an electric field.

FIGS. 40A-40B are graphs of electric field distribution and strengthwithin the liquid layer in response to photoconductor illumination.

FIG. 41 is a schematic of an experimental setup for trapping E. colicells with an OET device according to an aspect of the presentinvention.

FIGS. 42A-42B are images of cells being collected, “focused”, accordingto an aspect of the present invention.

FIGS. 43A-43B are images of cells being transported according to anaspect of the present invention.

FIG. 44 is a graph of cell movement speed in response to distance andoptical power according to an aspect of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring more specifically to the drawings, for illustrative purposesthe present invention is embodied in the apparatus generally shown inFIG. 1 through FIG. 44. It will be appreciated that the apparatus mayvary as to configuration and as to details of the parts, and that themethod may vary as to the specific steps and sequence, without departingfrom the basic concepts as disclosed herein.

The present invention includes numerous embodiments in which particles,cells, and other elements suspended in a fluid are manipulated. Theapplication describes these embodiment within nine sections.

1. Massively Parallel Manipulation Using Optical Images.

An optical image-driven dielectrophoresis technique is described hereinthat permits high-resolution patterning of electric fields on aphotoconductive surface for manipulating single particles. The techniquecan be performed at substantially lower light intensity levels than wererequired using previous techniques, for example one embodiment requiresapproximately 100,000 times less optical intensity than opticaltweezers. In addition, the technique can make use of incoherent lightsources. In one example an incoherent light source (a light emittingdiode (LED) or a halogen lamp) is utilized with a digital micromirrorspatial light modulator to demonstrate parallel manipulation of 15,000particle traps within a 1.3 mm×1 mm area. With direct optical imagingcontrol, multiple manipulation functions can be easily combined toachieve complex, multi-step manipulation protocols.

It has not been appreciated in the industry that optically-inducedelectrophoresis could be could be controlled with a dynamic opticaladdressing mechanism to provide the capability to perform manipulationdown to the single particle level. The optoelectronic tweezers (OET) ofthe present invention utilizes direct optical images to createhigh-resolution DEP electrodes for the parallel manipulation of singleparticles. DEP force results from the interaction of the induced dipolesin particles subjected to a non-uniform electric field. The magnitude ofthe force depends on the electric field gradient and the polarizabilityof the particle, which is dependent on the dielectric properties of theparticle and the surrounding medium.

FIG. 1 illustrates an example embodiment 10 of optoelectronic tweezers(OET) according to the present invention. Liquid 12 that containsmicroscopic particles (or cells) 14 is sandwiched between the upperlayer 16, such as comprising a transparent conductive ITO glass, and thebottom layer 18, such as comprising a photosensitive surface fabricatedfrom an ITO-coated glass 20 topped with multiple preferably featurelesslayers with 50 nm of heavily doped a-Si:H 22, 1 μm of undoped a-Si:H 24,and 20 nm of silicon nitride 26. By way of example the lower layer isshown upon a glass substrate 28.

The top 16 and bottom 18 surfaces are coupled to a bias source 30 suchas an AC electric signal of 10 V_(PP). Alternatively, the surfaces canbe less preferably DC biased, or biased with a combination of AC and DC,depending on the specific device structure and manipulation application.It should be appreciated that the frequency of the AC bias determineswhether particles, cells, and the like are attracted or repelled by thepatterned light, wherein the use of AC provides a number of advantagesover the use of a DC bias.

The photosensitive surface 24 of the lower layer converts the receivedoptical energy into a corresponding electric field, shown by way ofillustration by the set of concentric rings 32 of FIG. 1. Theillumination source may be any convenient light source, such as an LED34 operating at a wavelength of 625 nm (i.e., manufactured by Lumileds®,Luxeon® Star/O®) as depicted in this example. An optical projectionmeans provides a mechanism for outputting light patterns onto thephotosensitive lower layer 18, and preferably is configured foroutputting dynamic light patterns having spatial intensity variationover the surface, and typically structures defined by light or darkregions, which are output in a pattern stream (i.e., similar to amovie), or a pattern sequence (i.e., similar to a slide show). Onepreferred technique for projecting the light patterns is using a spatiallight modulation means 36, such as a digital micromirror display (DMD)which in combination with objective 38 focuses the light from LED 34onto the photosensitive surface 24 creating the non-uniform electricfield for DEP manipulation.

When projected light illuminates the photoconductive layer, it turns onthe virtual electrodes, creating non-uniform electric fields andenabling particle manipulation via DEP forces. These featureless layerscan be made without using any lithography or microfabrication, makingthe device inexpensive and attractive for disposable applications. TheOET-based optical manipulation has two operational modes, positive OETand negative OET, as a result of DEP forces induced for actuation.Particles can be attracted by or repelled from the illuminated area,depending on the AC electric field frequency and the internal andsurface dielectric properties of the particle.

As a consequence of the high photoconductive gain, the minimum opticalintensity required to turn on a virtual electrode is 10 nW/μm², which isapproximately 100,000 times lower than that of optical tweezers. Thislow threshold of optical intensity opens up the possibility of usingincoherent optical images to control the DEP forces over a large area,such as over a maximum area on the order of one square millimeter (1mm²), or even larger depending on optical configuration. For example,the optical images are created in one embodiment by combining an LED anda digital micromirror spatial light modulator (i.e., a DMD device suchas by Texas Instruments® having 1024×768 pixels with a 13.68 μm×13.68 μmpixel size). The pattern is imaged onto the photoconductive surfacethrough a 10× objective. The resulting pixel size of the virtualelectrode is 1.52 μm. The illumination source for the example was a redLED (625 nm wavelength) with 1 mW output power (measured after theobjective lens), which is sufficient to actuate 40,000 pixels. Tightfocusing is not required for OET, and the optical manipulation area canbe magnified by choosing appropriate objective lens. Using a 10×objective, the manipulation area (1.3 mm×1.0 mm) is 500 times largerthan that of optical tweezers.

The patterning of high-resolution virtual electrodes is critical forachieving single particle manipulation. OET has higher resolution thanthe optically-induced electrophoretic methods reported previously. Theminimum size of the virtual electrode is limited by the lateraldiffusion length of the photogenerated carriers in the photoconductor aswell as the optical diffraction of the objective lens. The large numberof electronic defect states in undoped a-Si:H results in a shortambipolar electron diffusion length of less than 115 nm. The ultimatevirtual electrode resolution is thus determined by the opticaldiffraction limit. In addition, the induced OET force is proportional tothe gradient of the square of the electric field, making it wellconfined to the local area of the virtual electrodes, which is also akey property for single particle manipulation.

It should be appreciated that the OET of FIG. 1 may be implemented witha number of variations according to the present invention, the followingbeing provided by way of example. The OET device is provided with afirst surface and second surface separated by chamber walls andconfigured for retaining a liquid which contains particles, or cells,being manipulated. It is preferred that electrodes are provided on thefirst and second surfaces of the OET upon which a biasing current and/orfield can be applied through and/or across the retained liquid. FIG. 1is shown with a single photoconductive surface. However, the presentsystem may be implemented having at least onephotosensitive/photoresponsive surface which induces a local electricfield on the surface of the material in response to received light,therein creating virtual electrodes for manipulating particles, cells,and the like at low optical power levels. It should be understood thatthe OET according to the invention may be created with a single ordouble-sided photosensitive surface, in various combinations with aconductive surface, non-conductive surface, or no opposing surface (openstructure).

The first and second surfaces of the OET are preferably formed as acontinuous film, wherein lithographic patterning of the surface is notnecessary for practicing the invention. However, it should beappreciated that the OET of the present invention can be implemented incombination with conventional DEP structures or continuous opticalelectrowetting techniques (COEW) toward specific application areas.

The OET of FIG. 1 can be configured having surfaces of amorphous and/ormicro/nano-crystalline semiconductor materials, amorphous Si, or organicphotoconductor materials, used with or without dielectric layers, suchas silicon nitride, silicon dioxide, and so forth. When used, the thindielectric layers of the OET, having an impedance that is much less thanthe impedance across the liquid retained in the OET device.

FIG. 2A through FIG. 2D illustrate results from an embodiment of thedevice which provides massively parallel manipulations of singleparticles across 15,000 particle traps created across a 1.3 mm×1.0 mmarea. The 4.5 μm diameter polystyrene beads experiencing negative DEPforces are trapped in the dark area.

FIG. 2A depicts a portion of the array, with each trap in thisparticular embodiment having a diameter of 4.5 μm to fit a singleparticle.

FIG. 2B illustrates by way of example parallel transporting of singleparticles with three snapshots from the captured video showing theparticle motion within this section of the manipulation area. Thetrapped particles in two adjacent columns move in opposite direction asseen in FIG. 2C and FIG. 2D. The induced negative DEP forces push thebeads into the non-illuminated regions, where the electric field isweaker. The size of each trap is optimized to capture a single 4.5 μmdiameter polystyrene bead.

By programming the projected images, these trapped particles can beindividually moved in parallel as shown in FIG. 2B. Compared with theprogrammable CMOS DEP chip, the particle trap density of the OET (11,500sites/mm²) is 30 times higher in response to the high-resolutionaddressing ability. Using direct imaging, sophisticated virtualelectrodes can be easily patterned and reconfigured to create dynamicelectric field distributions for continuous particle manipulationwithout the assistance of fluidic flow.

FIG. 3A through FIG. 3D illustrate an embodiment by way of example of anintegrated virtual optical machine in which the motion of differentcomponents is synchronized. In FIG. 3A, the image illustrates thestructure which integrates a number of virtual components including anoptical sorter path, conveyers, joints and a wedge. It should beappreciated that the OET device of the present invention can beconfigured for implementing a large variety of traps, combs, sorters,concentrators, loops, conveyers, joints, particle channels, wedges,sweepers, which can be implemented separately, in arrays of manipulationelements, and combinations and sequences of manipulation elements and soforth, without departing from the teachings of the present invention.

In FIGS. 3B-3C, two polystyrene particles with sizes of 10 μm and 24 μmpass through the sorter path and are fractionated in the z-direction dueto the asymmetry optical patterns. The particle traces can be switchedat the end of the sorter path by reconfiguring the tip position of theoptical wedge. The trajectories of particle movement are highlyrepeatable and accurately defined, as can be seen in FIG. 3B and FIG. 3Cin which the optical sorting repeatability is represented by the darkand light tracks. The light and dark loops in FIG. 3B represent theparticle traces after 43 cycles. The trace broadening at the checkingbar has a standard deviation of 0.5 μm for the 10 μm bead and 0.15 μmfor the 24 μm bead.

It should be appreciated that particles are transported throughdifferent functional areas and recycled in this light-patterned circuit,traveling through different paths depending on the position of the wedgedivider. Particles with different sizes are fractionated in the lateralz direction as they pass through the sorter path due to the asymmetricshape of the light-patterned electric fields. At the end of the sorterpath, an optical wedge divides and guides the particles into the twoconveyors. The looped optical conveyors recycle the particles back tothe sorter input to repeat the process.

FIG. 3D shows a distribution of particle position in the middle of thesorter (marked by a white bar) after the particles have passed throughthe sorter 43 times. The standard variations of trace broadening are 0.5μm for the 10 μm bead, and 0.15 μm for the 24 μm bead. The magnitude ofthe DEP force is proportional to the particle volume. The largerparticles exhibit tighter confinement in the optically patterned DEPcages during transport.

FIG. 4A through FIG. 4D illustrate by way of example the sorting ofbiological cells according to their characteristics. According to thisexample embodiment, the living cells are subject to positive OET,trapping them in the bright areas, and pulling the live cells into thecenter of the pattern. The dead cells (i.e., stained with Trypan Bluedye) leak out through the dark gaps and are not collected. By exploitingthe dielectric differences between different particles or cells, the DEPtechniques described herein have been used to discriminate and sortbiological cells with differences in membrane properties (e.g.,permeability, capacitance, conductivity, and so forth), internalconductivity, and cell sizes. It should be appreciated that thesetechniques can be extended to other particle or cell characteristics.The OET technique not only inherits these DEP advantages but alsoprovides the capability of addressing each individual cell.

The selective concentration of live human B cells is demonstrated from amixture of live and dead cells in FIG. 4A through FIG. 4D. The cells aresuspended in an isotonic buffer medium of 8.5% sucrose and 0.3%dextrose, mixed with a solution of 0.4% Trypan Blue dye to check thecell viability, resulting in a conductivity of 10 mS/m. The applied ACsignal is 14V_(PP) at a frequency of 120 kHz. The cell membranes of livecells are selectively permeable and can maintain an ion concentrationdifferential between the intracellular and extracellular environments.By contrast the dead cells are unable to maintain this differentialionic concentration difference. So, then dead cells are suspended in amedium with a low ion concentration, the ions inside the cell membraneare diluted through ion diffusion, which results in a difference betweenthe dielectric properties of live and dead cells. Live cells experiencepositive OET, and are collected in the center of the shrinking opticalring pattern by attraction to the illuminated region, while dead cellsexperience negative OET and are not collected.

Single cell analysis is an important technique to comprehend manybiological mechanisms since it is capable of determining the responsespectrum of each individual cell under stimulation. A new single celland particle manipulation technique has been demonstrated according tothe invention which has enabled manipulating a large number of singlecells and particles in parallel using direct incoherent optical images.By programming the projected optical patterns, multi-step diagnosticprotocols can be achieved by combining multiple functions such astransporting, sorting, recycling, and separating on a planar amorphoussilicon-coated glass slide. In addition to biological applications, thehigh resolution electric field patterned on an OET surface can alsoserve as a dynamic template to guide the crystallization of colloidalstructures.

2. Optoelectronic Tweezers.

The optoelectronic tweezers (OET) of the present invention are designedfor cell and microparticle manipulation using optical control to permitfunctions such as cell trapping, collecting, transporting, and sortingof cells and microparticles by using sequentially projected imagescontrolled by a spatial light modulator (microdisplay or DMD mirrors).Since the optical actuating power is as low as 1 mW, our inventionpermits the optical manipulation using a lightly focused incoherentlight source and a direct image projection system. The system providessubstantially increased optical manipulating area and allows thecreation of complex optical patterns and thus more optical manipulationfunctions.

FIG. 5 is a schematic example of the structure of an OET device 50according to another embodiment of the invention. The example OET shownincludes spacers 44, 46 defining the horizontal extent of the liquidcell structure and separating opposing surfaces 16, 18. An AC bias maybe applied across the top and bottom layers while optical patterns areimaged on a photosensitive surface within the lower layer 18. In apreferred embodiment the device comprises two opposing surfaces with atop surface having a transparent layer 16 with a thin conductive layer44 (i.e., aluminum) on an interior surface and a bottom layer 18 withphotosensitive surface 24. When the light is illuminated on thephotosensitive surface, such as comprising a layer of amorphous silicon,it creates a light defined virtual electrode 52 which generates anon-uniform electric field in the liquid layer 12. The cells orparticles nearby the virtual electrode are manipulated by theelectrostatic force.

FIG. 6 illustrates the OET of FIG. 5 within a cell manipulation system70. A programmable spatial light modulator 72, such as a programmableDMD, is used to generate the required optical image controlled by aprocessing means 74, such as a PC. The optical image is projected ontothe OET device, for example by reflecting light generated by a lightsource, such as halogen lamp 76, to create virtual electrodes within thestructure for cell manipulation.

The OET devices can be operated with or without pumps or channelsdepending on the desired applications. Optional liquid pump 78 andoutput ports 80 can be utilized for moving a liquid along with particlesor cells through the OET, or to change the conditions of the OET.

The motion of the cells is captured by an imaging means 88, such as acamera (i.e., charge coupled device (CCD)) with microscopic focus, whichprovides a feedback signal for further processing. In one mode of theinvention a magnification lens system 82 is coupled through acombination of color filters and beamsplitter 84 and a light source 86(i.e., mercury lamp) to allow images to be captured by computer 74.

When an optical image is projected onto the photosensitive surface, itcreates a light patterned virtual electrode as shown in FIG. 5. Thisvirtual electrode generates an electric field around it for manipulatingthe cells and particles through electrostatic force. The electric fieldpatterns generated by the optical patterned electrode can have any kindof shape depending on the image projected. It can form an electric fieldcage to capture a single cell, or cell groups, or form an electric fieldchannel to guide cells. Since those virtual electrodes are all opticallypatterned, they are fully programmable and reconfigurable. Opticalmanipulation functions such as cell transport, cell collecting and cellsorting can be achieved simultaneously on a single chip.

The exemplary OET device is particularly well-suited for applications incell manipulation at both multi-cell and single cell levels. Liquidcontaining cells (or particles) are first sandwiched between the twosurfaces of the OET. The optical images to be used are generated in thecomputer and then loaded for the spatial light modulator, which isilluminated with either a coherent or incoherent light source as shownin FIG. 6. The image is then preferably projected onto the OET devicethrough an objective lens to create the optical patterned virtualelectrodes. The response of the cells or the particles can be capturedby an imaging means (i.e., camera) as a feedback signal for the computerto generate a new optical pattern required for the optical manipulation.

The microscopic imaging means coupled to the OET is preferablyconfigured with recognition algorithms which provide information thatallows image patterns to be created based upon the number,characteristics, and position of the particles, and/or cells, retainedwithin the OET. For example this recognition algorithm can be utilizedfor determining particles and/or cells of specific size ranges, ofspecific colors and textures, or other directly detectablecharacteristics such as colors, shape, texture, conductivity,permeability, capacitance, motility, and so forth. The imaging system isalso preferably configured for detecting indirect characteristics suchas can be inferred from registering the response of particles, and/orcells, to environmental changes (e.g., aqueous solution changes,irradiation, temperature, pH, and so forth), or to interaction betweenthe particle, and/or cell, and other particles, cells, and/or structureswithin the OET.

It will be appreciated that characteristics of particles, or moretypically cells, can be determined by microscopically detecting responseto changes in the environment, such as a shift in color, shape, texture,and so forth of a particle or cell in response to a temperature change,irradiation change, chemical characteristics change of the surroundingliquid, interaction with other particles and/or cells, and so forth. Themicroscopic imaging means can be configured to store information foreach particle, or cell, in its field of view and to classifycharacteristics in response to correlating detected changes in responseto changes in the environment. The microscopic imaging means can retainthe information about each particle, or cell, despite its movementwithin the OET. In this way the present invention can be implemented toperform a wide range of particle and cell sorting, separation,classification, concentration, assembly, and other desired objects ofmanipulation.

The OET device can also be integrated with pumps and channel structuresto provide for continuous optical manipulation. Furthermore, the OETdevice can be combined with continuous optical electrowetting (COEW) orconventional fixed electrode DEP techniques to address specificapplications suited to a hybrid approach.

It should be appreciated that the present invention provides majorimprovements to the art with respect to the OET structure and in theutilization of photoconductive material. The OET device of the inventioncan be comprised of substantially featureless layers which do notrequire photolithography masking for fabrication, wherein fabricationcost factors are substantially reduced. In addition, it should beappreciated that low cost amorphous silicon is preferably utilized asthe photoconductive layer, while also providing the benefits of low darkconductivity, high photosensitivity and short electron diffusionlengths. It should be appreciated that aside from amorphous silicon,other materials with similar electrical and optical properties can alsobe utilized. In addition, alternate embodiments can be providing byusing other mechanisms and forms of photoresponsivity, such as using aphototransistor in place of the photoconductor structure.

The properties of the OET invention provide for optical manipulation atvery low optical power levels (i.e., on the order of 1 mW) andsub-micron resolution of virtual electrode and also permits opticalmanipulation with the OET using an incoherent light source. It will beappreciated that conventional OET devices rely on the use of coherentlight sources, as dictated by their structures.

The OET device of the present invention has been described and can beused for a variety of applications, such as particle trapping,collecting, multi-addressing, sorting on both microscopic particles andlive cells. The OET device of the invention allows for parallel opticalmanipulation of cells on both single and multi-cell levels usingreconfigurable optical patterns from a direct image projection system.No pumps, no microchannels and no valves are required to handle cells inmicrofluidic environment. It is contemplated that the use of theinventive OET device and methods described herein will provide asignificant step forward in the field of particle manipulation, and inparticular the manipulation of cellular particles.

3. Dynamic DMD-Driven Optoelectronics Tweezers for ParticleManipulation.

The ability to move and sort single cells is highly sought after in thebiomedical and biological community. Optical tweezers, and dynamicholographic optical tweezers (HOT) arrays have provided a means ofperforming individual cell manipulation, but require high optical powerlevels (approximately 1 μW-100 μW) and have a small trap area (<1 μm).Optoelectronics tweezers (OET) provides a method of cell manipulationwhich overcomes the shortcomings of optical tweezers. It requires verylow optical power (i.e., on the order of microwatts), which opens up thepossibility of using incoherent light source and direct optical imagingto pattern the traps.

Previously, we had demonstrated OET manipulation of microscopic latexspheres and live E. coli cells using a single laser beam. A spatiallight modulator can be used to generate multiple OET traps and novelpatterns such as line and ring cages. In this paper, we report on novelparticle cages capable of trapping and moving micro-particles by using adigital micromirror device (DMD) to project dynamic images onto our OETdevice, via a standard multimedia projector. It will be appreciated thatthis aspect of the invention demonstrates microscopic particlemanipulation using a non-coherent light source, which should providenumerous benefits within a number of applications.

FIG. 7A illustrates an example embodiment 90 of an optoelectronictweezers according to the invention which is based on the principle ofoptically-induced dielectrophoresis. A buffer solution 12, sandwichedbetween the nitride layer and the indium-tin-oxide (ITO) top layer,contains the particles 14 of interest. In operation a light source isfocused onto photoresponsive layer 24, such as comprising an AC-biasedamorphous silicon (a-Si) photoconductive substrate layer of the OETdevice. In the dark, the a-Si is highly resistive, however, as thephotoconductive layer is illuminated, the conductivity of the a-Si isgreatly increased, due to photogenerated charge carriers, to create alocalized virtual electrode, and generate a non-uniform electric fieldin the buffer solution. Dielectrophoretic (DEP) forces result from thenonuniformity of the electric field. These forces are either positive(particles attracted to electric field maxima) or negative (particlesattracted to electric field minima), depending upon the dielectricproperties of the particle and the media and the bias frequency. FIG. 7Billustrates an experimental setup for the OET material shown in FIG. 7A.

FIG. 8 depicts the spatial electric field distribution resulting from aring pattern projected onto the OET surface which can be configured toform a single-particle trap. Negative DEP forces hold a particle in thecenter of the light ring, as this corresponds to a local electric fieldminima. Particles outside the ring are repelled by the same forces.

Considering in detail the experimental setup 100 shown in FIG. 7B, shownby way of example, a computer 102 (i.e., a personal computer (PC))outputs image signals to an InFocus® LP335 DMD-based projector 104 usedas both the light source 114 (i.e., having a 120-W 1000-ANSI lumenhigh-pressure mercury lamp) and as the DMD driver circuit interface 116.The DMD, such as comprising an array of MEMS mirrors, forms an imagecorresponding to the output of the external monitor port of the PC.Light at the output of the projection lens was collected, collimated,and directed by way example with optics 118, lenses 106, 108, mirror 110into an objective lens 112 (i.e., 10×) onto OET device 90. The objectivefocused the beam into the buffer solution 12 with a conductivity of 0.1mS/m, sandwiched between the ITO top layer and photoconductive bottomlayer. The photoconductive layer was situated on the stage of a Nikon®TE2000E inverted microscope. Observations were made via a CCD cameracoupled into the inverted microscope.

FIGS. 9A-9F depict images of particle caging and trapping according toaspects of the present invention. The images in these figures wereformed on the focal plane of the objective using an optical projector,such as standard presentation software (Microsoft PowerPoint) on a PCconnected to the DMD projector. Negative DEP forces were observed on the25 μm latex spheres in solution, at an AC bias of 19.5V and a frequencyof 100 kHz. A variety of patterns were used to manipulate the particles,including dynamic line cages (FIGS. 9A-9C) and ring traps (FIGS. 9D-9F).Particle movement was observed to be approximately 40 μm/sec.

It should be appreciated that this aspect of the invention demonstratesmanipulation of micron-sized particles using optically-induceddielectrophoresis from a non-coherent light source. Various dynamiclight patterns were successfully used as particle traps andmanipulators, moving 25 μm latex spheres at approximately 40 μm/sec in a0.1 mS/m buffer solution.

4. Dynamic Array Manipulation of Particles Via Optoelectronic Tweezers.

Cellular-scale manipulation is an important tool in biological research,and technologies that have demonstrated the capability for suchmicroscopic manipulation include optical tweezers and dielectrophoresis.Although optical tweezers afford very fine control of microparticles,the technique suffers from high optical power requirements.Dielectrophoresis has been demonstrated to trap particles as small as 14nm. However, dielectrophoresis requires a static pattern of electrodes,and is not easily reconfigurable.

Accordingly, the present invention demonstrates another method ofmanipulating micrometer-scale objects using a technique ofoptically-induced dielectrophoresis, or optoelectronic tweezers. Using alaser to induce dielectrophoretic forces, the controlled movement of 25μm latex particles, and E. coli bacteria has been demonstrated. Thistechnique can be utilized at very low optical power levels, enabling themanipulation of particles and cells with an incoherent light source. Theuse of a spatial light modulator in the described optical system alsoallows for dynamic reconfiguration of particle traps, providingincreased versatility in particle manipulation over conventionaldielectrophoresis. The present invention describes novel manipulationaspects in which dynamic array manipulation of microparticles isperformed using optoelectronic tweezers. The self-organization ofparticles into an array, and the formation of single particle arrays,are demonstrated and provide the capability to individually address eachparticle.

Dielectrophoresis (DEP) refers to the forces induced upon a particle inthe presence of non-uniform electric fields, which are typicallygenerated by a variety of electrode configurations. A particle within anelectric field forms an induced dipole, which will experience a forcedue to the field gradient. The direction of the induceddielectrophoretic force is dependent upon the frequency of the electricfield and the permittivity and conductivity of the particle and thesurrounding medium. Positive DEP results in particle attraction toelectric field maxima. In contrast, negative DEP causes particles to berepelled from field maxima. Applying an AC electric field thus allowsthe tuning of the type of DEP force induced on a particle, as well asnegating any electrophoretic effects, or particle movement due to itssurface charge.

The optoelectronic tweezers (OET) device according to this aspect of theinvention enables optically induced dielectrophoresis. Unlikeconventional DEP, no electrode pattern is required to introducenon-uniformities into an applied electric field; instead, aphotoconductive layer is used to form virtual electrodes. Focusingincident light onto the photoconductor substantially increases itsconductivity as compared to the dark areas, effectively creating anelectrode in the illuminated area, analogous to the patterned electrodesin conventional DEP. In addition, the virtual electrodes used by OET aremovable and reconfigurable, unlike the static electrodes of conventionalDEP.

FIGS. 10A-10B illustrate aspects of an OET trap according to the presentinvention. In FIG. 10A, a single particle OET trap is shown with a 45 μmpolystyrene sphere contained by optically-induced negative DEP. In FIG.10B distribution of the square of the electric field is shown for thesingle particle trap in along the cross-section A-A′ as shown in FIG.10A. DEP force is proportional to the gradient of this distribution.

Shown by way of example, and not limitation, the single-particlerectangular trap of FIG. 10A has inner dimensions of approximately 70 μmby 50 μm. A sphere with a diameter of 45 μm is shown “captured” by thesurrounding light “walls” which are approximately 25 μm wide. Thecorresponding cross-sectional distribution of the square of the electricfield shows that width of the trap as experienced by the particle isapproximately 50 μm, as DEP force depends on the gradient of thisdistribution as shown in FIG. 10B. If negative DEP forces are induced bythe trap pattern, all particles outside the trap area will be repelledby the electric field maxima forming the trap perimeter. Any particlewithin the enclosed trap area will feel similar repulsive forces,however, these forces balance and trap the particle. Once a particle iscontained within the rectangular pattern, the trap can be moved,transporting the particle to a desired location. Furthermore, multipletraps can be used as building blocks to form arrays of trappedparticles, which can be arbitrarily arranged, and dynamicallyreconfigured.

The optical power required to induce DEP forces in the OET is much lowerthan that required when implementing optical tweezers, as the lightenergy provided for OET does not directly trap the particles. Earlyexperiments using OET showed movement of 25 μm particles at 4.5 μm/secwith an optical power of 1 μW, corresponding to an incident powerdensity of 440 mW/cm². In comparison, a 1 μm diameter optical tweezerstrap, at a minimum trapping power of 1 mW, has an optical power densityof 32 kW/cm².

The low optical power requirements of OET provide a number of systemdesign advantages. Inexpensive incoherent light sources can be employedinstead of lasers to provide the illumination necessary for OET. Inaddition, light patterns can be produced by imaging techniques (i.e.,raster or vector based) rather than scanning techniques. Furthermore,with no need to focus all optical energy, a simple spatial lightmodulator can be utilized to pattern images, rather than utilizing theholographic techniques employed by optical tweezers arrays.

FIG. 11 illustrates an embodiment 90 of the optoelectronic tweezersdevice with a liquid buffer containing the particles of interest betweenthe upper ITO glass layer and the lower photoconductive layer. Toseparate the top and bottom layers, 100 μm thick spacers (not shown) areutilized.

In demonstrating the OET device and methods herein the digitalmicromirror device (DMD) in a light projector was used to image thevirtual electrodes. An embodiment of the optoelectronic tweezers devicewas formed by evaporating a 10 nm thick aluminum film onto a glasssubstrate to provide electrical contact. A 1 μm thick undoped amorphoussilicon (a-Si) photoconductive layer was then deposited, for example byutilizing plasma-enhanced chemical vapor deposition. It should beappreciated that detailed fine-pitched features need not be created onthe first and second retention layers, wherein detailed lithographicsteps are not necessary. To protect the photoconductive film, a 20 nmthick silicon nitride layer is preferably deposited over the a-Si inthis embodiment. It should be appreciated, however, that for someapplications the device can be formed without a dielectric. The liquidbuffer layer containing the particles of interest is sandwiched betweenthis photoconductive device and the opposing surface, such as comprisingindium-tin oxide (ITO) glass. An applied AC bias across the ITO and a-Siproduces the electric field.

Amorphous silicon has a dark conductivity of about 0.01 μS/m to 1 μS/m.Thus, in the dark, the a-Si has a much lower conductivity than theliquid buffer (which has a conductivity of 10 mS/m), causing themajority of the voltage to drop across the silicon layer. Incident lightfocused onto the photoconductive layer substantially increasesconductivity and creates a non-uniform electric field surrounding theilluminated area, as the majority of the voltage now drops across theliquid buffer layer. In this manner, the light incident on the OETdevice can pattern virtual electrodes for dielectrophoresis.

FIG. 12 illustrates by way of example an experimental setup 100 for OET90. In this example embodiment, the image from a projector 104, such asan InFocus LP335, having mercury lamp 114, DMD 116, and focusing optics118 is focused via optical elements 106, 108 and 110 into a 10×objective lens 112, and projected down onto OET device 90. Particlemovement is observed on a microscopic imaging means 120, for example aNikon TE2000U inverted microscope, coupled to computer 102.

A DMD-based projector (InFocus LP335) is shown used to display imagesdrawn on a PC, via Microsoft PowerPoint software. The projector providesboth the optical source (a 120 W, 1000-ANSI lumen high-pressure mercurylamp) and the DMD-to-PC interface. The output of the projector iscollected, collimated, and directed into an objective lens (i.e.,Olympus MSPlan10 10X with NA=0.30), projecting an image onto the OETdevice. The power at the projector output was measured to beapproximately 600 mW.

Approximately 7% of this power is collected by the objective lens andfocused onto the OET device. Therefore, the power of the light incidenton the OET is 42 mW, corresponding to an intensity of 12 W/cm². Thebuffer solution comprises deionized water and KCL salt, mixed to obtaina conductivity of 10 mS/m. Polystyrene microspheres (45 μm and 20 μm)are mixed into the buffer solution, and sandwiched into the OET device.

FIG. 12 illustrates, by way of example, a optical setup embodiment 100for this OET demonstration. Observation of the particles under test isperformed preferably utilizing a microscopic imaging system 120, forexample a Nikon TE2000U inverted microscope. A CCD camera attached tothe observation port of the microscope recorded images and video ofthese demonstrations and tests. To produce the electric field necessaryfor DEP, an AC voltage of approximately 10V_(PP) at 100 kHz (i.e.,Agilent® 33120A) was applied across the top ITO surface and the bottomphotoconductive surface of the OET device.

FIGS. 13A-13B illustrate self-organization of 45 μm polystyrene spheresinto an array configuration. After the initial grid illumination shownin FIG. 13A, the randomly arranged particles move towards the dark areasvia negative DEP. After five seconds, all particles are contained withinthe array cells as shown in FIG. 13B.

The self-organization of randomly distributed 45 kHz polystyrene spheresinto an array (FIGS. 13A-13B) is demonstrated by directing a simple gridpattern of orthogonal horizontal and vertical lines, such as drawn inPowerPoint, which are projected onto the OET device. The patternactivates the optically-induced DEP, repelling particles from theilluminated areas due to negative DEP forces. This mechanism causes theself-organization of the particles once the grid pattern is illuminated;wherein particles are pushed into the non-illuminated cells. After asettling period, the particles are trapped within the array of cells.

Due to a large trap relative to the particle size, the initialself-organization may result in more than one particle per array cell asshown in FIG. 13B. In this array, the largest cells are 80 μm by 100 μm.It may be possible to form self-organizing arrays with a single particleper array cell by optimizing the dimensions of a single array cell trap,such that only one particle may fit into the potential well of the trapat any time.

FIGS. 14A-14B illustrate examples of single-particle manipulation withinthe array. A particle in the lower-left side of the array is made tochange its array by combining cells in FIG. 14A, the re-splitting thecell, moving the particle to the adjacent array position shown in FIG.14B.

In addition, it was found that certain particles within the selforganized array are able to escape when the array is moved around theimage plane. This phenomenon occurs for the array cells that containmultiple particles. This occurrence, along with subsequent manipulationof the self-organized array in FIG. 13B, allowed us to obtain an arraywith a single particle per cell as shown in FIG. 14A. It should be notedthat we were able to move the resulting array of single particles aroundthe image plane at approximately 25 μm/sec.

Particles can be moved individually between adjacent cells, asillustrated in FIGS. 14A-14B. The adjacent cells are merged by firstremoving the dividing wall, and then re-separating the cells. Allmovement of the trap walls are controlled in real-time by the operator.To improve on the speed of this technique, a moving light wall can beused to facilitate the transportation of the particle between cells.This enables a single particle to be transferred to any cell of thearray, using repeated transfers between adjacent cells.

FIGS. 15A-15D illustrate examples of flushing an array row to removeundesired particles from the array. First, the walls of the cells in therow to be flushed are removed as shown in the top row of FIG. 15A. Theparticles are no longer bounded in the lateral direction as shown inFIG. 15B. An operator-controlled light bar is then used to push theparticles out of the array as shown in FIG. 15C and FIG. 15D.

Since the patterns for manipulating the particles in the array arecreated dynamically by optical illumination, a wide variety ofoperations can be performed by simple software programming. For example,to flush the particles in a single row of the array, we remove thedividing walls of that row and use a moving wall to sweep out theparticles (FIGS. 15A-15D).

In addition to self-organizing behavior, arrays can be formed frommultiple single-particle traps. Each randomly positioned particle isfirst contained within a square trap. This is performed by drawing arectangle around each particle in PowerPoint. The multiple traps canthen be positioned to form an array of individually addressable cells.

FIG. 16 illustrates an example of an array of single particles, formedfrom multiple single particle square traps. Each particle isindividually addressable. The time required to form this array of 20particles was 3 minutes. Using this technique, we are able to form a 4×5array of single particle traps as shown in FIG. 16. Though the operationwas performed manually, it can potentially be automated by combining OETwith a vision system. Biological applications of such an array includestudies on single-cell behavior and interaction. Since each cell of thearray is an independent single particle trap, the array has thecapability of being dynamically rearranged.

FIGS. 17A-17D illustrate an example of dynamic rearrangement of an arraycontaining both 45 μm and 20 μm particles. An array is rearranged bymoving individual cells into a desired configuration. Totalrearrangement time for the embodiments was three minutes. The spheresare reorganized under operator control in the images shown in FIGS.17A-17D, which demonstrates the addressability of each particle trap, aswell as the dynamic nature of the OET patterns.

Movement of a single 45 μm sphere in response to negative DEP provides amaximum velocity of approximately 35 μm/sec. This corresponds to anestimated force of 15 pN, based upon Stoke's Law. The maximum velocityof a 20-particle array is limited to approximately 25 μm/sec. Thus, theminimum holding force of each individual array cell is 10 pN. This forceis less than that experienced by a single 45 μm particle, probably dueto slight nonuniformities in image sharpness over the entire array area.The more defocused areas will have less of an electric field gradient,and a correspondingly lower DEP force. Thus, this 10 pN force reflectsthe minimum trapping force of all of the array cells.

The forces attained in these experiments, using an optical power densityof 12 W/cm², are in rough agreement with our earlier results using a 632nm laser light source. Our earlier data suggests that the optical powerdensity necessary to achieve a force of 15 pN is 6.6 W/cm². Thedifference between this predicted power density requirement and ourexperimental findings can be attributed to losses through the additionaloptics needed for our current experiment.

These results compare favorably to other microparticle manipulationtechniques. Conventional dielectrophoresis uses static electrodepatterns, and is thus not reconfigurable. In addition, our device isless expensive to produce, as no photolithographic steps are needed.Addressable DEP arrays have also been demonstrated using CMOStechnology, but these devices are expensive to produce, and the minimumelectrode size is limited by the required CMOS circuits. Bothconventional DEP and optical tweezers are capable of manipulatingparticles a few nanometers in diameter. The minimum size of the virtualelectrode in OET is limited by the 115 nm ambipolar diffusion length ofthe a-Si. The OET can operate over a large area (i.e., approximately 1×1mm), which is much greater than the 20 μm×20 μm area for opticaltweezers.

Though holographic tweezers can generate multiple traps, direct imagingusing a DMD is more versatile. It can generate any arbitrary patternwith high contrast ratio. No computation is required to generate thedesired pattern. Furthermore, OET can induce repulsive forces ontransparent dielectric particles such as biological cells, and form cellcages, which is not possible with optical tweezers. On the other hand,optical tweezers traps are three-dimensional, whereas our trap patternsare limited to two dimensions. Utilization of the present inventiongenerally requires being more selective in the choice of buffersolutions, because the conductivity of the solution plays an importantrole in the DEP phenomenon.

The self-organizing of 45 μm polystyrene particles into an array, andthe creation of an array from multiple single particle traps utilizingoptoelectronics tweezers have been demonstrated with the presentinvention. Single particle movement within the array has beendemonstrated, showing the ability to address individual array cells.Movement of single 45 μm polystyrene spheres was measured to be 35μm/sec (a force of 15 pN). Movement of a 20-particle array was performedat 25 μm/sec (a force of 10 pN). Such particle manipulation techniqueshave many applications to experiments with biological cells andmicroparticles.

5. Microvision-Activated Automatic Optical Manipulator for MicroscopicParticles.

An embodiment of the present invention includes an automatic opticalmanipulator that integrates microvision-based pattern recognition andoptoelectronic tweezers (OET) for processing microscopic particles. Thissystem automatically recognizes the positions and sizes of randomlydistributed particles and creates direct image patterns to trap andtransport the selected particles to form a predetermined pattern. Byintegrating the OET with a programmable digital micromirror devicedisplay (DMD), we are able to generate 0.8 million pixels of virtualelectrodes over an effective area of 1.3 mm×1 mm. Each virtual electrodeis individually controllable for parallel manipulation of a large numberof microscopic particles. Combining the automatic microvision analysistechnology with the powerful optical manipulator, this systemsignificantly increases functionality and reduces processing time formicroparticle manipulation.

Tools for manipulating microscopic particles are important in the fieldsof cell biology and colloidal science. Optical tweezers anddielectrophoresis are two of the most widely used mechanisms formanipulating microparticles. Optical tweezers use direct optical forcesto deflect the motion of microscopic particles. Optical tweezers arenoninvasive and have high positioning accuracy. The use of holographicoptical tweezers further extend the benefits to allow manipulatingmultiple particles. However, these techniques require very high opticalpower levels, and provides limited working area (<100 μm×100 μm) due tothe need of tight focusing with high numerical aperture (N.A.) lenses.These factors limit the use of these forms of optical tweezers inlarge-scale parallel manipulation applications.

In contrast, dielectrophoresis (DEP) controls particle motion bysubjecting particles to non-uniform electric fields. The techniqueprovides high throughput and large working area, but requires a fixedelectrode pattern for a given function. Programmable DEP cage arrayconsisting of two-dimensional electrodes with integrated drivingcircuits has been demonstrated on a CMOS (complementarymetal-oxide-semiconductor) chip. However, the resolution is limited bythe pitch of the electrode and the driving circuits of the unit cell,and the cost may prohibit its use as disposable devices.

FIGS. 18A-18B illustrate aspects of an example embodiment of an opticalmanipulation system. In FIG. 18A a schematic diagram is shown of anexample embodiment 130 of a microvision-based automatic opticalmanipulation system. In FIG. 18B the structure of the OET device isshown.

According to the present invention a novel optoelectronic tweezers (OET)has been developed to address DEP forces on a photoconductive surfaceusing optical beams. OET enables virtual electrode patterns to becreated optically. The electrode size can be varied continuously by theoptical spot size down to the diffraction limit of the objective lens.Because of the optoelectronic gain in the photoconductor, the requiredoptical power density is five orders of magnitude lower than that ofoptical tweezers. This enables our method to use a digital opticalproject with incoherent light source to manipulate microparticles. Thepresent invention describes the use of “light walls” to confinemicroparticles in virtual microfluidic channels and switch them by lightpistons. Interactive manipulation of virtual DEP cage arrays has alsobeen demonstrated by manually changing the optical patterns.

In this aspect of the invention automatic optical manipulator use isdescribed by integrating the OET with a microvision-based analysissystem. The microvision system automatically recognizes the particlepositions and sizes, generates the desired trapping patterns, andcalculates the moving paths of the particles. It enables close-loopcontrol of trapping, transporting, and assembling a large number ofparticles in parallel.

The microvision based optical manipulation system 130 of FIG. 18A isconstructed with OET device incorporating a microscopic imaging meansand a mechanism for registering particle/cell characteristics andposition.

Particle or cell movement is controlled by projecting light generatedfrom source 114 reflected from DMD 116 through objective 112 onto OET90. The light patterns are generated in response to the positioning andcharacteristics of the particles or cells as registered by a microscopicimaging means in combination with image analysis and pattern recognitionalgorithms. In this example the images are collected through lens 132onto a CCD imager 134, and the data is processed to control the patternsof light being generated. The microscopic images, or image stream, isanalyzed within an image analysis circuit and/or routine 136. The imagedata is then processed using pattern recognition circuits and/orroutines 138. The recognition of actual patterns is performed inrelation to the desired goal of the application, wherefrom subsequentpatterns are generated by pattern generator circuit and/or routine 140,which is then converted by DMD circuit and/or routine 142 to control theoperation of the programmable DMD device 116. It should be appreciatedthat this may be implemented in a number of alternative ways withoutdeparting from the teachings of the present invention, such as usingvarious imaging sources, microscopic imaging, and different techniquesfor detecting, analyzing, and generating subsequent optical images ontothe OET.

By way of example, this embodiment incorporates a Nikon invertedmicroscope 132, 134. A 150 W halogen lamp 114 illuminates on aprogrammable digital micromirror device (DMD) microdisplay 116. The DMDpattern is imaged onto the OET device through a 10× objective lens 112.The structure of OET device 150 is shown in FIG. 18B.

FIG. 18B illustrates another example OET embodiment comprising a top andbottom surface 16, 28, comprising such as indium-tin-oxide (ITO) glassand photosensitive layer 24, such as amorphous silicon, on the surfaceof the top and/or bottom layers. The liquid medium 12 containing theparticles 14 are sandwiched between these two surfaces. The OET isbiased by a single AC voltage source 30. Without light illumination,most of the voltage drops across the amorphous silicon layer 24 becauseits impedance is substantially higher than liquid layer 12. Underoptical illumination, the conductivity of the amorphous Si 24 increasesin the areas upon which the illumination is impinging by several ordersof magnitude, shifting the voltage drop to the liquid layer. Thislight-induced virtual electrode thereby creates a non-uniform electricfield 152, and the resulting DEP forces drive the particles of interest.The light-induced DEP force can be positive or negative, controlled bythe frequency of the applied AC signal. Negative DEP force repelsparticles away from the high field region, and is preferable for singleparticle cage, which can be easily formed by a light wall around theparticle. Positive DEP tends to attracts multiple particles. We haveemployed negative DEP force in our automatic optical manipulatorexperiments. The image on the OET device is captured by a CCD camerathrough the inverted microscope and sent to a computer for imageprocessing.

Software according to the present invention analyzes the real time videoframes and generates the corresponding optical patterns for trapping andmoving the particles. These patterns are then transferred to the DMD,and our test setup allows direct control of individual pixels. Theresolution of the projected optical image on the OET device is 1.3 μm,defined by the pixel size of the mirror (13 μm). The effective opticalmanipulation area on the OET is 1.3 mm×1 mm. By combining the DMDmirrors with the OET device, the silicon-coated glass is turned into amillion-pixel optical manipulator.

FIGS. 19A-19D illustrate the process of automatically recognizing andarranging randomly distributed particles into a predetermined pattern.First, the images of the particles are captured and analyzed by themicrovision system as in FIG. 19A, which identifies the positions andthe sizes of all particles as in FIG. 19B. The software then generates aring trap around each particle as in FIG. 19C. It also calculates thetrajectories of the particles to reach their final positions as in FIG.19D.

FIGS. 20A-20D illustrate by way of example test images for a particlerecognition system. Polystyrene particles with three different sizes, 10μm, 16 μm, and 20 μm, are mixed and randomly distributed in the liquidmedium. In FIG. 20B, the microvision system recognizes the position ofeach particle and projects a ring mark on each particle. In FIG. 20C,the histogram showing the number of particles versus the number ofrecognized dark pixels in this test image. In FIG. 20D the largestparticles are selectively picked up by setting a threshold for the darkpixels.

Particle recognition is achieved by using a dark-pixel recognitionalgorithm to scan through each pixel of the captured image. Thebrightness value and the position of each pixel are then recorded andcalculated to determine the size of each particle and its centerposition. The brightness value of the pixels at the particle edge issmaller than that of the background and the color is darker too. Bysetting a threshold brightness value between the background and theparticle edge, we can recognize the edge pixels of each particle.Averaging the x and y position data of the edge pixels of each particle,we can determine its position.

FIG. 20B shows the recognized particles marked by a white ring patterngenerated by the microvision analysis system. The same algorithm alsodetermines the size of each particle by counting the number of therecognized dark pixels.

FIG. 20C is a histogram of data showing the number of particles and thenumber of the dark pixels recognized for each particle on this image. Aslarger particles have more dark pixels than smaller ones, a thresholdnumber can be set for the recognized pixels, as indicated by the dashline in the histogram figure, wherein the system can selectivelyregister particles with certain sizes.

FIG. 20D depicts the seven largest beads (20 μm), by way of example,that are selected by setting a threshold number equal to 180. Thisrecognition algorithm is used specifically for determining sphericalparticles with different sizes. Other algorithms can be developed torecognize particles with different colors, shapes, or textures.

FIGS. 21A-21B illustrate, by way of example, the electric fielddistribution induced by a single optical ring pattern. In static stateshown in FIG. 21A the particle is trapped in the electric field minimumin the center. During moving as shown in FIG. 21B the particle isdisplaced from the center as a result of the balance between the DEP andthe viscous forces.

Particle trapped by an optical ring pattern trapping of a singleparticle is achieved by operating OET in the negative DEP regime. Wecreate an optical ring pattern to form a virtual DEP cage that allowsonly one single particle to be trapped inside the ring, as shown in FIG.21A.

In static state, the trapped particle will be focused at the center orthe ring pattern where the minimum electric field strength occurs. Whenthe optical ring moves, the trapped particle also move in the samedirection but with a position deviated from the ring center so that theDEP force pushes the particle in the direction toward the center. Thisdeviation distance depends on how fast the particle moves. When theoptical ring moves too fast, the particle will escape the optical ringbecause the DEP force is not strong enough to hold it. The escapingspeed of a 20 μm particle is 40 μm/sec in our current system. To trap aparticle with a smaller size, a smaller optical ring would be requiredto ensure a single particle in the ring.

FIGS. 22A-22C each illustrate multi-frame example sequences ofmicrovision-based automatic optical manipulation of microscopicparticles. In FIG. 22A randomly distributed particles are shown beingarranged into a hexagonal shape. In FIG. 22B the hexagonal pattern ofFIG. 22A is shown being transformed into a line. In FIG. 22C the linepattern of FIG. 22B is then transformed into a triangle shape. In eachcase the unwanted particles are swept away by a scanning line.

Once the particle positions are recognized, the software is configuredto generate the corresponding ring-shaped traps and calculates thetransport trace for each particle. These optical patterns are stored asimage files and are batch loaded to the DMD control software to createdynamic optical patterns to trap and transport particles. Theseprocesses are shown in FIG. 22A. The image of the randomly distributedparticles was scanned vertically from left to right. The first sixparticles were identified and trapped by the OET by the 0 second frame.The trapped particles were transported by moving the ring traps, andreached the hexagonal configuration in 12 seconds.

FIGS. 22B and 22C show the video sequences of rearranging the particlesinto linear and triangular shapes and the unwanted particles were sweptaway by a scanning line pattern.

An automatic optical manipulator has been demonstrated that provides afeedback control through a microvision analysis system. This system canautomatically recognize particles with specific size from a mixture ofparticles with different sizes and generate optical manipulatingpatterns to trap and move these selected particles to form apredetermined pattern. The large optical manipulation area (>1 mm×1 mm)of our OET device permits parallel manipulation of a large number ofmicroscopic particles. The automatic parallel optical manipulationsystem greatly reduces the time for sorting and patterning microscopicparticles. With further optimization, the system will be able to sortparticles with different colors, shapes, or textures. More sophisticatedoptical manipulation functions can also be performed. The automaticoptical manipulator has many potential applications in biological cellanalysis and colloid science fields.

6. Manipulation of Live Red and White Blood Cells.

Optoelectronic tweezers (OET) provides a new tool for single-cellmanipulation for biological research applications. Currentcell-manipulation technologies, such as optical tweezers anddielectrophoresis, have limitations that can be overcome by OET.

Optical tweezers are a widely used tool for the manipulation of cellsand microparticles in the micro-scale and nanoscale regimes. Byintegrating holographic imaging techniques with optical tweezers,multiple particle traps can be created from a single laser source.However, optical tweezers requires expensive, high-power lasers, and islimited in its effective manipulation area.

Dielectrophoresis (DEP) describes induced particle motion along anelectric field gradient due to the interaction of the induced dipole inthe particles and the applied electric field. This technique has beenused to perform many biological experiments, including cell and DNAtrapping and cell sorting. A limitation of conventional DEP devices isthe difficulty of reconfiguring the devices for different experiments,as they rely on patterned metal electrodes to create the requirednon-uniform electric fields. By using CMOS technology to create DEPtraps, real-time reconfigurable DEP devices can be achieved. However,these CMOS-based devices have a limited resolution, due to the area ofthe circuitry.

Optoelectronic tweezers offer low-power optically-controlled actuationof cells and microparticles via light-patterned virtual electrodes on aphotoconductive surface. Since OET is directly controlled by opticalimages, it is easy to reconfigure in real time. In addition,high-resolution cell manipulation is achieved over a large area. As oneof the major potential applications of OET is biological analysis, anumber of experiments were performed using live cells. The firstdemonstration of OET on living cells was performed by Chiou et al., onE. coli bacteria, proving the low optical power of OET is capable ofmanipulation of live single cells without causing photodamage. In thisaspect of the invention we present a description and demonstration ofOET manipulation of live mammalian cells.

Optical tweezers work by directly converting photon momentum to amechanical force on a microparticle or nanoparticle. This requires ahighly-focused, intense laser beam. In contrast, optoelectronic tweezerscreates an optically-patterned electric field, which in turn produces adielectrophoretic force on particles. Due to the photoconductive gain,the required optical power is on the order of 100,000 times less thanthat required of a typical optical tweezers. As a result, an incoherentlight source, such as an LED or halogen lamp, is sufficient for OETactuation. Furthermore, the optical pattern does not need to be highlyfocused, allowing OET to be effective over a larger area (currently 1.3mm×1.0 mm) than optical tweezers traps.

FIG. 23 illustrates an example embodiment 170 of the structure of an OETdevice according to the invention. The OET consists of an upper planarelectrode 16 of ITO-glass and a lower photoconductive layer 24, betweenwhich are sandwiched a layer of liquid solution 12 containing the cellsor microparticles 14 of interest. The upper planar electrode in thisembodiment consists of a conductor such as indium-tin-oxide (ITO) whichitself is transparent over a transparent glass slide, while the lowerphotoconductive layer is preferably formed with hydrogenated amorphoussilicon (a-Si:H) deposited onto a ITO-coated glass slide viaplasma-enhanced chemical vapor deposition (PECVD). Projecting lightpatterns 172, 174 modifies the electric field profile, creating adielectrophoretic force. An AC bias 30 is placed across the upperelectrode and the lower photoconductive layer.

In the dark regions, the applied AC voltage is dropped primarily acrossthe highly-resistive (R_(S)) a-Si:H layer, which has a much higherimpedance than (R_(L)) of the liquid 12, resulting in a low electricfield in the liquid solution. However, in the illuminated regions, theprojected light creates virtual electrodes, by locally increasing theconductivity of the a-Si:H. The photoconductor is now less resistivethan the liquid, creating a high electric field region in the liquidabove the virtual electrode. This creates non-uniform electric fields,which in turn creates a dielectrophoretic force to drive the cells.

Dielectrophoretic force is AC frequency-dependent. Thus, by varying thefrequency of the applied AC bias, the force can be adjusted from anattractive force to a repulsive force, or vice-versa. Since OET usesoptically-induced DEP, the OET force is also tunable in the same manner.As a result, there are two operating modes for OET: positive OET, inwhich cells and microparticles are attracted to the illuminated areas,and negative OET, in which cells and microparticles are repelled by theilluminated areas.

FIG. 24 illustrates an example embodiment 190 configured formanipulation of bovine red blood cells utilizing optoelectronic tweezersaccording to the present invention. For this demonstration, the opticalsource 114 consisted of a 0.8 mW He—Ne laser (λ=633 nm). A spatial lightmodulator 116 is not necessary if a laser is used as the light source,as the laser can be directed into the objective either directly orthrough a mirror. A 10× objective lens 112 was used to reduce the laserbeam size to about 20 μm in diameter. A personal computer (PC) 102 isshown coupled to control the light imaging via laser output control or aspatial modulator as well as to control the signals generated at OETdevice 170, such as bias voltage, from function generator 192 and forcollecting data from a microscopic imager 88. The prepared cellsolutions consisted of red blood cells (RBCs) from bovine serum,suspended in an isotonic solution (8.5% sucrose, 0.3% dextrose) atconcentrations ranging from approximately 1 to 10% by volume.Approximately 5 μL of this solution was introduced into the OET device.

FIGS. 25A-25D depict resultant concentrations of blood cells using theOET of FIG. 24. A strong positive OET response was observed at anapplied AC bias of 3 V_(PP) at 200 kHz, attracting the red blood cellstowards the laser spot shown in FIG. 25A. Initially, the laser is on inFIG. 25A, but no electric field is applied. An AC bias is then appliedto the OET device, producing OET force, which attracts the blood cellsto the illuminated area as shown in FIG. 25B. It was also observed thatthe cells align vertically along the electric field lines as in FIG.25B. When the laser is turned off, the concentrated cells remain in thearea that the laser spot was focused as shown in FIG. 25C. As theapplied voltage is switched off, the concentrated red blood cells beganto slowly pulsate, migrating away from the central area as shown in FIG.25D, implying that they remain alive and viable.

The use of an incoherent light source and direct image patterningtechniques increases the flexibility and functionalities of OET. Aspatial light modulator can pattern any arbitrary image to be projectedonto the photoconductive surface, creating the corresponding virtualelectrodes on the OET device. Complex, reconfigurable manipulationpatterns can thus be created by simple software programming. Thispowerful technique is demonstrated in the arrangement of humanB-lymphocytes into a complex pattern.

A 100 W halogen lamp was used as the incoherent optical source. Thespatial light modulator consisted of the Texas Instruments digitalmicromirror device (DMD). The DMD is a 1024×768 array ofindividually-addressable micromirrors, each of which is 13.68 μm×13.68μm. The images displayed on the DMD are controlled via a computer. A 10×objective lens was used to increase the resolution of each DMD mirror toapproximately 1.4 μm. The prepared cell solutions consisted of humanwhite blood cells (B-lymphocytes), suspended in an isotonic solution.Approximately 5 μL of this solution was introduced into the OET device.

FIGS. 26A and 26B illustrate how B-lymphocytes can be manipulated intoan arbitrary pattern. In this example we chose to assemble the cellsinto the shape of a “U” (FIG. 26A) and a “C” (FIG. 26B) character (forUniv. of California). At an applied bias of 14V_(PP) at a frequency of100 kHz, the white blood cells exhibit positive OET behavior. Ashrinking concentric ring pattern is used to concentrate the cellstowards the character image. Cells are attracted to each concentricring. As the rings shrink, the cells are transported towards the centerof the concentric rings, where the character image is projected. Thecells then become trapped by the static character pattern.

Optoelectronic tweezers provides a powerful tool for single-cellmanipulation. The use of direct imaging and incoherent light sourcesprovides OET with more flexibility than conventional DEP. The OETtechnique also uses considerably less optical power than opticaltweezers, while still providing a larger effective manipulation area.Concentration and manipulation of cells, specifically red blood cells,has been demonstrated by using the OET and methods of the presentinventive aspect. It should be appreciated that these manipulationfunctions can be easily tailored to a specific biological experiment.

7. Novel Optoelectronic Tweezers Using Light Induced Dielectrophoresis.

Optical tweezers have become an important tool in biological researchareas since they were first demonstrated. However, the potentialphotodamage caused by the intense optical energy has restricted its use.For example, a 100 mW optical tweezers has a light intensity on theorder of 10¹⁰ mW/cm² when focused to diffraction limit. Such an intenselight energy may cause damages due to local heating or two-photonabsorption. To reduce the photodamage, lasers with wavelengths in thenear infrared region are often chosen to avoid the absorption in wateror biological objects. However, the recent research shows the cellmetabolism may still be affected even using infrared lasers.

Recently, a light induced electrophoresis mechanism has been proposed tooptically address polymer beads by using DC electric bias. Theelectrically charged particles are attracted to the electrode withopposite polarity. In this paper, we present a light induceddielectrophoresis mechanism that would allow the optical addressing ofelectrically neutral micro-particles with μW optical energy, which ismuch lower than the approximately 1 mW to 100 mW of optical energy usedby optical tweezers. Dielectrophoresis (DEP) refers the motion of anelectrically neutral particle resulting from the interaction between theapplied electric field and the induced dipole. It has been used widelyin the manipulation of microparticles or sub-micro-particles andbiological cells. An analytical expression of DEP force is given by thefollowing expression.

F_(dep) = 2 π a³ε_(m)Re[K*(ω)]▽(E²)${{K\text{*}(\omega)} = \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} - {2\; ɛ_{m}^{*}}}},{ɛ_{p}^{*} = {ɛ_{p} - {j\frac{\sigma_{p}}{\omega}}}},{ɛ_{m}^{*} = {ɛ_{m} - {j\frac{\sigma_{m}}{\omega}}}}$

According to the above equation E is field strength, a is particleradius, ∈_(m) and ∈_(p) are the permittivities of the surrounding mediumand the particle, respectively, σ_(m) and σ_(p) are the conductivity ofthe medium and the particle, respectively, with ω the angular frequencyof the applied electric field.

FIGS. 27A-27B illustrate an OET embodiment 210 in FIG. 27A with anIllustration of the light induced dielectrophoresis mechanism in FIG.27B.

The term Re[K*(ω)] can have any value between 1 to −½, depending on theapplied AC frequency and the polarizability of the particle and themedium. If Re[K*(ω)]<0, it is called negative DEP with the direction ofthe DEP force towards lower electric field. Since the DEP force isproportional to the gradient of the square of the applied electricfield, a highly non-uniform electric field is desired to achieve ahigher trapping force. In the following experiment a light inducednegative DEP force is demonstrated.

In FIG. 27A, the structure of the optoelectronic tweezers are shown witha liquid solution 12 containing the particles sandwiched between twosurfaces separated by a gap spacing of 100 μm. The top surface 16 is acommercial ITO glass. The bottom surface is a glass substrate 28 coatedwith three pattern-less layers: a 2000 Å-thick aluminum layer 46, a 2μm-thick photoconductive (amorphous silicon) layer 24, and a 200-Å-thicksilicon nitride layer 26. An AC bias 30 is applied between the top (ITO)and the bottom (aluminum) electrodes. In the dark state, the majority ofthe voltage drops across the photoconductor due to its high electricalimpedance, which results in a very weak electric field in the liquidlayer. When the laser beam 40 is focused through objective 38 onphotoconductive layer 24, the local photoconductivity at the site underlight illumination is greatly increased due to the photogeneratedelectron-hole pairs.

FIG. 27B depicts a light defined micro electrode turned-on locally andcreating a highly non-uniform field in liquid layer 12. The laser spotcreates a light defined electrode and a highly non-uniform electricfield in the liquid layer. The particles inside the liquid are polarizedby the non-uniform field and pushed away from the illuminated site bythe negative DEP force.

Since light is used to switch the AC voltage drop between thephotoconductive layer and the liquid layer, rather than to directly trapthe particles, the required optical power is orders of magnitude lowerthan that of conventional optical tweezers.

FIG. 28 depicts experimental results for the OET of FIG. 27A with therelationship between particle speed and optical power. In theexperiment, a 800 μW laser with a beam width 0.24 mm and a wavelength of632 nm is used as the light source. The laser beam is preferably steeredby a pair of orthogonally scanning galvanometer mirrors and then sentthrough a combination of a convex lens and a 40× objective lens. Theoptical spot size on the photoconductive layer is around 17 μm. Neutraldensity filters are used to control the incident optical energy. A 100kHz AC bias is applied between the top and the bottom electrodes todrive 25 μm latex particles. To measure the particle speed, the scanningmirror is programmed to scan at a constant speed to push the particle.The particle is pushed by the optical beam, until at sufficiently highscan rate, the particle can no longer keep up with the optical beam. Themaximum speed at which the particle responds to scanning optical beam ismeasured for various optical powers and AC bias voltages. An opticalbeam with power as low as 1 μW light is sufficient to transport theparticle at a speed of 4.5 μm/sec at 10 V AC bias. The maximum speedobserved here is 397 μm/sec, which corresponds to a force of 187 pNestimated by Stokes' law.

FIG. 29A and FIG. 29B illustrate an example of multi-particle focusing,in which the laser beam is programmed to scan in circular patterns. Thefour particles are focused, or squeezed, to the center of the shrinkingcircular pattern.

According to the present aspect of the invention a novel optoelectronictweezers is demonstrated which is successfully applied to transportneutral micro particles. The required optical power on the order of from(i.e., approximately 1 μW-100 μW) is one to two orders of magnitudeslower that that of optical tweezers. Particle transport speed of 397μm/sec and trapping force of 187 pN are measured for 25 μm latexparticles with 100 μW optical power and 10 V AC bias.

8. Optical Sorting Mechanism in Dynamic Electric Field.

Optoelectronic tweezers (OET) have been proposed herein as a powerfultool for cell and microparticle manipulation, via direct optical images.The optically-patterned electrical field generated on the OET surfacecan be configured to trap and transport single or multiple cells inparallel. Such a dynamic reconfigurable electric field provides drivingforces for sorting particles without the need for pumps to introducefluid flow, as presented in most of the microfluidic sorters. Itcompletely eliminated the need for the fabrication and integration ofcomplex microfluidic components, adding lots of flexibility in theapplications of cell or particle manipulation. In the present inventionan OET based sorting mechanism is demonstrated using a dynamic movinglight beam. Particles on the OET surface can be sorted simply byscanning a light beam across the OET surface. The sorted particles canbe transported to other areas by other dynamic optical patterns thathave been demonstrated in the industry. The following portion of thepresent invention focuses on the fundamental mechanism of OET-basedoptical sorting.

FIGS. 30A-30C illustrate dynamic electric fields induced by an OETaccording to the present invention. In FIG. 30A a schematic diagram isshown for the OET device, in which different sized particles 14, 14′,14″ are sandwiched between a top ITO glass 16 and a bottom OETphotoresponsive surface 24. In FIGS. 30B-30C randomly distributedparticles are sorted out when the line-shape laser beam scans across theOET surface.

Optoelectronic tweezers are a novel mechanism that enables opticalpatterns to induce highly non-uniform electric fields on aphotoconductive thin film material. The particles near the non-uniformelectric field experience a net force, resulting from the interactionbetween the electric field and the induced electric dipole of theparticles. This force is called dielectrophoretic (DEP) force, which canbe expressed in the following relation.

F_(dep) = 2 π r³ε_(m)Re[K*(ω)]▽(E²)

According to the above equation E is field strength, r is particleradius, ∈_(m) and ∈_(p) are the permittivities of the surrounding mediumand the particle, respectively, σ_(m) and σ_(p) are the conductivity ofthe medium and the particle, respectively, with ω the angular frequencyof the applied electric field and K*(ω) is the Clausius-Mossotti (CM)factor, which has a value between 1 and −0.5, representing thepolarizability of the particle.

This force is very sensitive to the size of the particle a³ and thenon-uniformity of the field ( VE²). If a particle is less polarizablethan the medium, its real part of the CM factor is negative, and theparticle will be pushed away from the high electric field area. When aline-shaped laser beam scans across the OET surface, it produces anelectric field pattern that moves at the same speed. This light-inducedelectric field will push particles in the OET device. The relativedistance between the moving light beam and the particles is determinedby the balance between the DEP force and the viscous force. UsingStoke's Law to estimate the viscous force for a moving particle, weobtain the following relationship between the particle size andnonuniformity of the field.

${r^{2}{\bigtriangledown\left( E^{2} \right)}} = {C = \frac{3\;\eta\; v}{ɛ_{m}{{Re}\left\lbrack {k\text{*}(\omega)} \right\rbrack}}}$

In the above equation r is the particle radius and C is a constantdetermined by the light scanning speed v, real part of the CM factor,and also the viscosity η, and permittivity ∈_(m), of the surroundingmedium. Since the term VE² is a function of the relative distancebetween the particle and the electric field maximum, particles withdifferent sizes will have different deterministic relative distances tothe center of the scanning laser beam. Based on this principle,particles with different sizes will be sorted out when the laser beamscans across the OET surface.

FIG. 31 illustrates an example embodiment 230 of an experimental setupfor optical sorting of microscopic particles. A single-mode fiberpigtailed laser diode 114 with a wavelength of 635 nm is coupled througha fiber collimator 232, producing a beam spot size of 3 mm and anoptical power of 120 μW. A cylindrical lens 234 directed through a 2Dscanning mirror 236 and dichroic mirror 238 and a 10× objective 38 lensare used to shape the circular Gaussian beam into a line shaped patternand focus it onto the OET surface. A scanning mirror is programmed tosteer the laser beam. The OET device 210 is shown on a stage 240 andconfigured with a microscopic imaging means for registering particle (orcell) position and characteristics within the OET.

FIGS. 32A-32D depicts the result when the laser beam (120 μW red diodelaser at wavelength=635 nm) scans across the OET surface of FIG. 31where the 10 μm and 20 μm diameter polystyrene beads are randomlydistributed. In FIG. 32A the particles are randomly distributed on thesurface. In FIG. 32B the optical beam scans across the area of the 10 μmbeads and aligns them into a line pattern. In FIG. 32C the 10 μm and 20μm beads are aligned and moving with different relative distances to thecenter of the optical beam. In FIG. 32D the optical beam is programmedto “jump” into the spacing between these two groups of particles andfurther separate them.

After the line-shaped laser beam scans across the assortment of beads,the 10 μm and 20 μm beads become aligned at different distances relativeto the center of the beam. The laser beam is programmed to “jump”between these two groups of particles and further separate them. Thissorting process finished in 25 seconds.

FIGS. 33A-33B shows the sorting of particles of different sizes;specifically 5 μm, 10 μm, and 20 μm particles with relative distances of15 μm, 20 μm, and 40 μm, respectively, under the scan speed of 6 μm/sec.The optical beam scans at a constant rate from the left to the right.These three sizes of particles are moving at the same speed as the lightbeam. Their deterministic relative distances remain constant during themovement. The relative distance is scan speed dependent. At a high scanspeed, the particle experiences a larger viscous force. In order tobalance this force, the particle moves closer to the scanning beam,where a stronger electric field gradient exists.

FIG. 34 depicts the relationship between the scan speed and the relativedistances of microparticles from the scanning beam center as a functionof scanning speed. Theoretical calculations are shown in solid lines andexperimental data is shown by the dots.

For the 20 μm particle, the relative distance to the scanning beamcenter decreases from 50 μm to 25 μm when the scan speed increase from20 μm/s to 100 μm/s. This trend is also reflected in the data for 5 μmand 10 μm particles. Thus, low scanning rates provide a larger spatialseparation between different sizes of particles. At a scanning speed of17 μm/sec, the spacing between 5 μm and 10 μm particles is 7 μm, and thespacing between 10 μm and 20 μm particles is 28 μm. The maximum scanningrate for a 5 μm particle is approximately 70 μm/sec. If the scanningrate is increased beyond this limit, the particle becomes levitated bythe vertical non-uniformity of the electric field, causing the particleto escape the lateral “pushing force” of the scanning beam. The escapespeed is higher for bigger particles; for a 10 μm bead, it is 90 μm/sec.

The present invention provides a novel optical sorting mechanism basedon optoelectronic tweezers (OET). The light induced dynamic electricfields sort out particles with diameters of 5 μm, 10 μm, and 20 μm bysimply scanning a light beam across the OET surface. This techniquecompletely eliminates the requirement of extra pumps as a driving forcefor liquid flow, greatly simply the fabrication and integration processof microfluidic system. The deterministic relative distances from thebeads to the beam center are size-dependent. Particle sorting performedon 5 μm and 10 μm-diameter beads resulted in a spacing of 7 μm betweenthe separated groups. The spacing between sorted 10 μm and 20 μmdiameter particles was 28 μm.

9. Moving Toward an all Optical Lab-on-a-Chip System.

Miniaturization and integration of microfluidic systems could reduce thecost as well as increase the speed of many analytical biological andchemical processes. Multiple microfluidic functions are integrated on achip, referred to as “lab-on-a-chip”, to perform the biologicalanalysis. These functions include microfluid delivery mixing, celltrapping, concentrating, and sorting. Conventional lab-on-a-chip systemsconsist of micro pumps, valves, and fluidic channels.

The fluidic circuits, and therefore their functions, are usually fixedby the specific structure which has been fabricated. By contrast to theconventional “fixed” microfluidic system, the optical manipulationapproach taught herein offers several advantages. The present inventionis flexible and easily re-configurable. Optical tweezers have beenwidely used to trap cells and other bio-particles, and recently,holographic optical tweezers have been proposed to perform multiparticletrapping, optical sorting, particle spinning, three-dimensionalmanipulation, and optical pumping of microfluid. These microfluidicfunctions permit an all-optical-lab-on-a-microscope system that isprogrammable and reconfigurable in response to light input. However, theoptical tweezers-based systems suffer from the following limitations.First, control of microfluids using optical force is not energyefficient. The optical energy is first transferred to the kinetic energyof colloidal particles or beads. The moving beads induce liquid flowthrough the viscous force. The maximum force from optical trap is around100 pN, which is not large enough to drive liquids through microchannelseffectively due to a large pressure drop. Second, the optical powerrequired by optical tweezers is very high. It requires tightly focusedlaser beams to provide optical gradient force for trapping or deflectingthe particle motion.

Typically, a single trap requires 1 mW of optical power, and multipletraps require even higher power. Here, instead of using optical forcethe present invention relies upon a light-induced electrowettingmechanism, called optoelectrowetting (OEW), for controlling microfluidsand a light-induced dielectrophoresis mechanism, called optoelectronictweezers (OET), for manipulating microscopic particles.

FIG. 35 illustrates the concept by a comparison of energy transfer pathsof different optical manipulation methods, wherein optical energy isfirst converted to electrical energy, which in turn drives the liquidsor particles through electrowetting or DEP processes, respectively. As aconsequence of the optoelectronic gain of the photoconductor, therequired optical power is reduced by four to five orders of magnitude.Optical tweezers transfer energy from optical domain directly tomechanical domain, while optoelectrowetting (OEW) and optoelectronictweezers (OET) transfer optical energy to electrical domain first andthen trigger the electrical force for the manipulation.

Surface tension is the dominant force for controlling liquids inmicroscale. Several mechanisms have been proposed to control the surfacetension. Electrowetting is attractive because of its fast response andlow power consumption. It changes the contact angle of a droplet on asolid surface by modulating the surface energy at the liquid-solidinterface with electrostatic energy. Optoelectrowetting uses opticalbeams to control the amount of electrostatic energy stored in thatinterface and thus the contact angle.

FIGS. 36A-36B illustrate an embodiment 250 of optoelectrowetting inwhich a water droplet 252 is placed on a glass substrate coated with atransparent conductive glass 20, a photoconductive layer 24, and a thindielectric layer 16. FIG. 36A depicts the droplet without lightillumination, with the contact angle 254 being the same as the initialangle without bias. In FIG. 36B illumination is generated by source 212and the contact angle of droplet 252′ decreases due to theelectrowetting effect.

An AC electrical bias is applied between the bottom electrode and thedroplet. The photoconductor is configured with a high electricalresistance in the dark, resulting in a RC charging time much longer thanthe AC signal cycle. A very small amount of the voltage drops across thecapacitor between the droplet and the photoconductor. The contact anglein the dark is the same as the initial angle without bias, as shown inFIG. 36A. When light shines on the photoconductor layer, it createselectron-hole pairs and increases the photoconductivity by severalorders of magnitude. The RC time becomes much smaller than the cycle ofthe AC signal, resulting in a fully charged capacitor, as shown in FIG.36B. These extra electrical charges stored in the capacitor change thesurface energy between the solid-liquid interface and thus the dropletcontact angel. The relation between the contact angel and the voltageacross the insulator can be expressed by the following.

${\cos\left\lbrack {\theta(V)} \right\rbrack} = {{\cos\left\lbrack \theta_{0} \right\rbrack} + {\frac{1}{2}\frac{ɛ}{d\;\gamma_{LV}}V^{2}}}$

In the equation above the value θ₀ is the initial contact angle, ∈ isthe permittivity of the insulator, d is the thickness of the insulator,γ_(LV) is the surface tension of the liquid-vapor interface, V is theroot-mean-square voltage of the applied signal.

FIGS. 37A-37C illustrate induced movement by electrowetting. In FIG.37A, a schematic of an example embodiment is illustrated which inducesmovement of a liquid droplet by an optical beam on the COEW surface. InFIG. 37B an equivalent circuit of the COEW device of FIG. 37A is shown.In FIG. 37C the layered structure of the COEW surface is shown.

OEW allows optical tuning of the voltage across the insulator and thusthe contact angle. Our previous results have demonstrated that usinghalogen lamp with an intensity of 65 mW/cm² is sufficient to reduce thecontact angle of the droplet from 105° to 75°, turning hydrophobicsurface to hydrophilic. Using OEW, we have demonstrated a droplet-basedmicrofluidic device that allows an optical beam to extract a dropletfrom a liquid reservoir and transport it freely on a two-dimensionalsurface. Separation of the droplet is achieved with two optical beamsmoving in opposite directions. The droplet (100 nL volume) follows thescanning optical beam up to a speed of 70 mm/sec, demonstrating theeffectiveness of OEW for optical manipulation of microfluid. The minimumdroplet size is limited by the area of the OEW electrodes (>1 nL for anelectrode area of 100 μm×100 μm).

For manipulating sub-nano-liter droplet the present invention includes acontinuous OEW (COEW) device that allows optical beams to create virtualelectrodes that can be continuously addressed on a two-dimensionalsurface.

FIG. 37A shows the structure of the COEW device. It consists of twosurfaces, a top ITO glass and a bottom photosensitive COEW surface. Thetop ITO glass is coated with a 0.2 μm thick silicon dioxide layer and a20 nm thick Teflon layer, making the surface hydrophobic; and the bottomCOEW surface consists of multiple featureless layers, including a 200 nmthick ITO, 10 nm thick aluminum, 5 μm thick undoped amorphous silicon,200 nm thick silicon dioxide, and a 20 nm thick Teflon layers, as shownin FIG. 37C. The liquid droplet is sandwiched between these two surfaceswith a 10 μm gap defined by a photoresist spacer. Due to the hydrophobicTeflon coating, the initial contact angle of the liquid-solid interfaceis larger than 90°. When a light beam illuminates at one edge of thedroplet, it creates a virtual electrode at the photoconductive layerright underneath this edge. The optoelectrowetting effect is turned onlocally, reducing the droplet contact angle at the illumination site.

This process can be understood from the equivalent circuit model shownin FIG. 37B. The amorphous silicon layer has smaller capacitance (higherAC impedance) than the silicon oxide layers in the dark because it isten times thicker. Very small amount of voltage drops across the siliconoxide layer. Under light illumination, the conductivity of amorphoussilicon increases by several orders of magnitude, thereby reducing theelectrical impedance to a much smaller value than that of the oxidelayers. The contact angle is reduced locally, creating an unbalancedpressure on the droplet. The net capillary force pushes the droplet tomove toward the laser beam. By scanning the light beam, the droplet iscontinuously addressed on the COEW surface. There are two factors thatmay limit the resolution of the light-patterned virtual electrodes:optical diffraction limit and ambipolar electron-hole diffusion length.In the case of amorphous silicon, the ambipolar diffusion length is lessthan 115 nm, resulting in a electrode resolution only limited by opticaldiffraction.

The movement of a 100 μL droplet moving on the COEW device is capturedby a video camera through a microscope. The movement is directed by a100 μW HeNe laser with a wavelength of 632 nm. The focused spot size is20 μm using a 10× objective. The laser beam is steered by a pair oforthogonal galvanometer scanning mirrors (Cambridge Inc.). A 100V ACbias with frequency of 10 kHz is applied through the top ITO glass andthe bottom COEW surface.

FIGS. 38A-38D show a sequence of video snapshots showing the dropletmoving in a circular pattern with approximately a 100 μm radius. Thespeed of the droplet is 785 μm/sec. These images show microdroplettransport by COEW. The droplet has a volume of 100 μL and moves in acircular pattern directed by a scanning laser beam. The speed of thedroplet is 785 μm/sec.

The virtual electrode created by light illumination can also be used tomove microscopic particles in liquid through dielectrophoretic (DEP)force. The DEP force is generated by the interaction of the appliedelectric field and the induced electric dipoles in neutral particles. Anaspect of the invention provides an optoelectronic tweezers (OET) devicethat exploits such light-induced DEP to move microscopic particles withvery low power optical beams. The DEP force has been widely used tomanipulate microscale or nanoscale particles. The analytic expression ofthe DEP force is given by the following equation.

F_(dep) = 2 π a³ε_(m)Re[K*(ω)]▽(E²)${{K\text{*}(\omega)} = \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} - {2\; ɛ_{m}^{*}}}},{ɛ_{p}^{*} = {ɛ_{p} - {j\frac{\sigma_{p}}{\omega}}}},{ɛ_{m}^{*} = {ɛ_{m} - {j\frac{\sigma_{m}}{\omega}}}}$

In the above equation E is field strength, a is particle radius. ∈_(m)and ∈_(p) are the permittivities of the surrounding medium and theparticle, respectively, σ_(m) and σ_(p) are the conductivity of themedium and the particle, respectively, with ω the angular frequency ofthe applied electric field. The value K*(ω) is the Clausius-Mossottifactor and is a frequency dependent complex number. The real part ofK*(ω), or Re[K*(ω)], has a value between 1 and −0.5, depending on thepolarizabilities of the medium and the particle and on the frequency ofthe applied AC electric bias. If Re[K*(ω)]>0, the particle will movetowards higher electric field region and this is called positive DEP. Onthe other hand, if Re[K*(ω)]<0, the particle will move away from thehigh field region and this is called negative DEP.

FIG. 39 illustrates an example embodiment of an OET structure in whichthe liquid containing the cells or particles are sandwiched between anITO glass and a photoconductive surface. To achieve light-induced DEP,we use a device structure that is very similar to the OEW device butwithout the Teflon and silicon dioxide layers on either the ITO or thephotosensitive surfaces, as shown in the figure. The 1 μm thickamorphous silicon is coated with 20 nm silicon nitride layer to preventelectrolysis. As in the OEW device, the amorphous silicon layer has highresistance in the dark, resulting a small voltage drop across the liquidlayer. Under light illumination, the virtual electrodes create anon-uniform electric field in the liquid layer, producing a DEP force onthe particles nearby. The particles can be attracted or repelled by theoptical beam, depending on the sign of the Clausius-Mossotti factor. Thephotoconductive gain of the amorphous silicon allows OET to operate withvery low optical power.

Our previous results showed a 1 μW He—Ne laser with wavelength at 633 nmis sufficient to transport a 25 μm particle at 4.5 μm/sec. Though thestructures are similar, the OET and the OEW are different in thefollowing aspect: the OET switches the voltage between thephotoconductive layer and the liquid layer, while the OEW switches thevoltage between the insulating layer and the photoconductive layer.

In this section, we present the applications of OET for collecting andtransporting biological cells. To accommodate the size of E. coli cells,the gap spacing in OET is reduced to 15 μm. FIG. 40A shows the simulatedelectric field distribution in the liquid layer for a 17 μm virtualelectrode generated by a focused laser beam and a bias voltage of 10 V.The conductivity of the liquid is 1 mS/m. The conductivity of theamorphous silicon follows the intensity distribution of the laser beam,and is assumed to have a Gaussian shape with a peak conductivity of 10mS/m at the center. The three-dimensional electrical field distributionis calculated using FEM-LAB.

FIG. 40A and FIG. 40B illustrate electric field properties for aphotoconductor layer. FIG. 40A illustrates electric field distributionin the liquid layer when the photoconductor is illuminated by a focusedlaser beam with 17 μm spot size. FIG. 40B The electric field strength atthree different heights above the photoconductive layer. The electricfield distribution at 4 μm, 8 μm, and 12 μm above the photoconductivesurface are plotted in FIG. 40B. Since the DEP force is proportional tothe gradient of E2, the electric field distribution shows that the OETcan generate strong DEP force within a radius of approximately 20 μm inthe lateral direction. The vertical gradient attracts the particlestowards the photoconductive surface. Both the lateral and the verticalgradients are strongest near the edge of the laser spot, similar tothose generated by a physical electrode.

FIG. 41 illustrates a demonstration setup for trapping biological cells.The OET device is coupled to an microscopic imaging means, such asplaced on an inverted microscope (i.e., Nikon® TE2000E) with thephotosensitive side up. A 0.8 mW He—Ne laser (wavelength=632 nm) is usedto power the optoelectronic tweezers. The incident power is controlledby neutral density filters. The optical beam is delivered to the devicethrough a 40× objective lens with a numerical aperture (N.A.) of 0.5,thereby producing a 17 μm focused spot size. The fluorescent image ofthe cells is captured by a CCD camera through the bottom objective lens.

FIG. 42A is an image of fluorescent E. coli cells before OET is turn on.FIG. 42B is the same image as FIG. 42A after the OET is turned on for 14seconds. It should be appreciated that the E. coli cells are “focused”by the OET to the laser spot.

When the laser beam is focused on a fixed spot, the OET attracts cellswithin the trapping area towards the center of the beam, as shown inFIG. 42A and FIG. 42B. It functions as a cell concentrator. In thisexperiment, we use the E. coli cells that can express green fluorescentprotein (GFP) for the convenience of observation under fluorescentmicroscope. The liquid has a conductivity of 1 mS/m. We apply a 100 kHz,10V_(PP) (volts peak-to-peak) AC electric bias between the top and thebottom ITO electrodes. The E. coli cells experience positive DEP forceunder these conditions. The effective capturing distance is around 20 μmfrom the focal point. Due to the electric field gradient in the verticaldirection, the cells are trapped right on top of the photosensitivesurface. When the laser beam is turned off, these trapped E. coli cellsswim away. No “opticution” is observed even for light in the visiblewavelength range, thanks to the low optical intensity.

We have investigated the minimum optical power required to operate thisOET. Cell concentrating is observed for optical power as low as 8 μW.This optical power density is almost five orders of magnitude lower thanthat of conventional optical tweezers with 1 mW laser focused todiffraction-limited spot size. The concentrated cells can be transportedto any arbitrary location by scanning the laser beam. FIG. 43 shows thetransport of multiple E. coli cells using a single scanning laser beam.

To study the effective trapping area and the velocity of the trappedcells, we recorded the trapping action and analyzed the video imagesframe by frame. The recording microscope is focused on the surface ofthe photoconductor to capture the trapped cells. We have measured thevelocities of cells trapped by lasers with optical powers of μm 8 μW,120 μW, 400 μW, and 800 μW. The OET traps work at all power levels.

FIG. 44 shows the measured velocities of the E. Coli cells towards thecenter of the focused light spot cells versus the radial distance fromthe center of the trap. At 800 μW, cells as far as 30 μm away areattracted by the OET. Initially, they move at a relatively low speed of5 μm/sec. The speed increases sharply when they are within 20 μm,eventually reaching a speed of 120 μm/sec at about 15 μm from the focalpoint. The transport speed becomes smaller after the peak value. Thecells are stopped at 9 μm from the center by the trapped cells. Thisresult matches very well with the simulated electric field distributionin FIGS. 40A-40B. The maximum slope of the top curve (4 μm abovephotoconductor) happens at about 15 μm from the center. The cellvelocity is a function of the optical power. The peak velocity increasesfrom 26 μm/sec at 8 μW to about 90 μm/sec at 120 μW. Above 120 μW, thepeak velocity increases more slowly, and eventually saturates at about200 μW. This can be explained by the following: when the optical poweris lower than 120 μW, the photoconductor is not fully turned on, forexample the conductivity is lower than, but not negligible, compared tothe liquid. At about 200 μW, the conductivity of liquid becomes dominantin the electrical circuit, and most of the electric field drops acrossthe liquid layer.

Further increases of optical power do not change the peak electricfield. However, the electric field distribution becomes more “square”like because of this saturation effect. The capturing area increasesslightly after the peak field saturates. It should be pointed out thatthe minimum optical power required for OET depends on the liquidconductivity.

The liquid conductivity used in our experiments is 1 mS/m. The currentlaser power can attract cells in liquid with conductivities up to 100mS/m. The optical power can be further reduced by shrinking the opticalspot size. The current power level can be reduced by 100 times bydecreasing the spot size from 17 μm to 1.7 μm.

In this present aspect of the invention we have presentedoptoelectrowetting (OEW) and optoelectronic tweezers (OET), formanipulating microdroplets and microparticles by light. The OEW useslight-induced electrowetting to control the surface tension, thedominating force in microscale, and actuate microdroplets. Our resultshows that a 100 pL water droplet is transported at a speed of 785μm/sec with an optical power of 100 μW. The OET exploits light-induceddielectrophoretic force for manipulating microparticles. The opticalpower required by OET is as low as 8 μW and the optical power density isfive orders of magnitude lower than that of optical tweezers. We haveused OET to concentrate and transport live E. coli cells withoutphotodamage. The low power requirement of OET opens up the possibilityof trapping microscopic particles using incoherent light sources.

Embodiments have been described for practicing the apparatus and methodof the invention by way of example. It should be appreciated that thespecific demonstration/experimental setups were provided only by way ofreference and that the invention can be implemented in a wide variety ofways using various equipment as will be recognized by one of ordinaryskill in the art. It should be recognized that although the presentinvention provides an OET which can be implemented with unpatternedsurfaces, the teachings herein can be combined with patterned techniquesto provide a hybrid approach without departing from the teachings of thepresent invention. Additionally, specific values for particle transport,times, and other measured characteristics were provided to aid those inunderstanding the approximate results which can be gleaned from thistechnology; one of ordinary skill in the art will appreciate that inmany cases the results can be significantly improved with more detailedimplementations beyond these demonstrations. Furthermore, it should beappreciated that the aspects of the present invention can be practicedon the areas as described, or in other areas which will be recognized bythose of ordinary skill in the art based on the teachings herein.

Although the description above contains many details, these should notbe construed as limiting the scope of the invention but as merelyproviding illustrations of some of the presently preferred embodimentsof this invention. Therefore, it will be appreciated that the scope ofthe present invention fully encompasses other embodiments which maybecome obvious to those skilled in the art, and that the scope of thepresent invention is accordingly to be limited by nothing other than theappended claims, in which reference to an element in the singular is notintended to mean “one and only one” unless explicitly so stated, butrather “one or more.” All structural, chemical, and functionalequivalents to the elements of the above-described preferred embodimentthat are known to those of ordinary skill in the art are expresslyincorporated herein by reference and are intended to be encompassed bythe present claims. Moreover, it is not necessary for a device or methodto address each and every problem sought to be solved by the presentinvention, for it to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed under the provisions of 35U.S.C. 112, sixth paragraph, unless the element is expressly recitedusing the phrase “means for”.

What is claimed is:
 1. An apparatus for manipulating cells or particlesby light induced dielectrophoresis (DEP), the apparatus comprising: afirst surface and a second surface configured for retaining a liquidcomprising particles or cells to be manipulated; at least onephotoconductive area on said first or said second surface configured forconversion of received light to a local electric field in the vicinityof the received light; a light source to provide the light received bythe photoconductive area; wherein the local electric field selectivelyrepels or attracts particles or cells; a microvision-based patternrecognition subsystem which is configured for controlling the output ofsaid light source in response to registering the position of, andoptionally the characteristics of, particles or cells as determined frommicroscopic imaging.
 2. An apparatus as recited in claim 1, wherein saidcharacteristics are selected from the group of particle and cellcharacteristics consisting essentially of size, color, shape, texture,viability, motility, conductivity, permeability, capacitance andresponse to changes in the environment of the particle or cell.
 3. Anapparatus for manipulating cells and particles using opticalimage-driven light induced dielectrophoresis (DEP) over atwo-dimensional area, comprising: a first surface and second surfaceconfigured for retaining a liquid containing particles, or cells to bemanipulated; at least one photoconductive area on said first or secondsurface which is configured for inducing a local electric field, virtualelectrode, in the vicinity of received light; an optical projector orscanning laser configured for generating dynamic sequentialtwo-dimensional light patterns onto said photosensitive surface therebyinducing dynamic localized electric fields for DEP manipulation ofparticles or cells; and a microscopic imaging subsystem which isconfigured for controlling the output of said optical projector inresponse to registering the position of, and optionally thecharacteristics of, particles or cells as determined from analyzingmicroscopic images.
 4. An apparatus as recited in claim 3, wherein saidcharacteristics are selected from the group of particle and cellcharacteristics consisting essentially of size, color, shape, texture,viability, motility, conductivity, permeability, capacitance andresponse to changes in the environment of the particle or cell.
 5. Anapparatus as recited in claim 1, wherein said microvision-based patternrecognition subsystem is further configured for controlling said lightsource to project a pattern of light onto said photoconductive area thattraps ones of said particles or cells.
 6. An apparatus as recited inclaim 5, wherein said pattern of light comprises traps each of whichtraps an individual one of said particles or cells.
 7. An apparatus asrecited in claim 6, wherein each of ones of said traps comprises anenclosure light pattern enclosing an individual one of said particles orcells, said enclosure light pattern creating an electric field cage thatrepels by dielectrophoresis said particle or cell, thereby trapping saidparticle or cell.
 8. An apparatus as recited in claim 7, wherein saidenclosure light pattern is a ring.
 9. An apparatus as recited in claim6, wherein said microvision-based pattern recognition subsystem isfurther configured for controlling said light source to change saidpattern of light projected onto said photoconductive area to move onesof said traps, thereby moving ones of said particles or cells.
 10. Anapparatus as recited in claim 3, wherein said microscopic imagingsubsystem is further configured for controlling said optical projectoror scanning laser to generate a pattern of light onto saidphotoconductive area that traps ones of said particles or cells.
 11. Anapparatus as recited in claim 10, wherein said pattern of lightcomprises traps each of which traps an individual one of said particlesor cells.
 12. An apparatus as recited in claim 11, wherein each of onesof said traps comprises an enclosure light pattern enclosing anindividual one of said particles or cells, said enclosure light patterncreating an electric field cage that repels by dielectrophoresis saidparticle or cell, thereby trapping said particle or cell.
 13. Anapparatus as recited in claim 12, wherein said enclosure light patternis a ring.
 14. An apparatus as recited in claim 11, wherein saidmicroscopic imaging system is further configured for controlling saidoptical projector or scanning laser to change said pattern of lightgenerated onto said photoconductive area to move ones of said traps,thereby moving ones of said particles or cells.